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ay Oa ie
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tak JOURNAL

EXPERIMENTAL ZOOLOGY

EDITED BY

WILLIAM K. BROOKS
Johns Hopkins University
WILLIAM E. CASTLE
Harvard University
EDWIN G. CONKLIN
University of Pennsylvania
CHARLES B. DAVENPORT
Carnegie Institution
ROSS G. HARRISON
Yale University
HERBERT S. JENNINGS
Johns Hopkins University

FRANK R. LILLIE
University of Chicago
JACQUES LOEB
University of California
THOMAS H. MORGAN
Columbia University
GEORGE H. PARKER
Harvard University
CHARLES O. WHITMAN
University of Chicago
EDMUND B. WILSON
Columbia University

ROSS G. HARRISON, Managing Editor
2 HILLHOUSE AVENUE, NEW HAVEN, CONN.

VOLUME V

PUBLISHED QUARTERLY BY
THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY
36th STREET AND WOODLAND AVENUE
PHILADELPHIA, PA,

CONTENTS

No. 1—November, 1907

Davin Day WHITNEY

Determination of Sex in Hydatina senta

ArTHUR B. Lams
A New Explanation of the Mechanics of Mitosis. With Two Figures... .

HERBERT EUGENE WALTER
The Reactions of Planarians to Light. With Fourteen Figures... .

No. 2—December, 1907

HERBERT EUGENE WALTER

The Reactions of Planarians to Light. With Fourteen Figures (con-
chided) pete Sy lsterie ce wo eta MEMES ora CR eae eee

Mary ISABELLE STEELE

Regeneration in Compound Eyes of Crustacea. With Sixteen Plates and
De Mane WOR endian mace edcae bdeauoheb hor sorgoesse onan

H. V. WiLson

On Some Phenomena of Coalescence and Regeneration in Sponges.

ANS NI SOME MN ateS a hace boo manbeu ss sco un bob aw ou oaU pmaOal dick

Hans PrzIpRAM
Equilibrium of Animal Form. With) Templo reson rerescaheteeretieeeteha (ter

CHARLES ZELENY

The Effect of Degree of Injury, Successive Injury and Functional Activity
upon Regeneration in the Scyphomedusan, Cassiopea xamachana.

\WitchvBourphiipuresssteilt qesicteece tty sa te cletmetstteret teeters Oot acy ett
ALEXANDER PETRUNKEVITCH
Studies in Adaptation. I. The Sense of Sight in Spiders. With Six
INANE, Foc pone ee Ree ah: SE Sk

245

259

No. 3—March, 1908
Gruman A. Drew

The Physiology of the Nervous System of the Razor-Shell Clam (Ensis
directus, Cons)) sWathi@neiPlate see cen: enecace.o = ote rer 311

FLORENCE PEEBLES

The Influence of Grafting on the Polarity of Tubularia. With Twenty-
Chay writs choeoss Guid bod O An ou 6 DMbore iD ROME ad oso. vac 32

N. M. STEvENS

A Study of the Germ Cells of Certain Diptera, with Reference to the
Heterochromosomes and the Phenomena of Synapsis. With Four

Pl atesi hire cei uakeee iS cphane ence BRS eea: ADE he kee Sus 9, ed Sie ee Re ere 359

Ratpu S. LILiie
Momentary Elevation of Temperature as a Means of Producing Artificial
Parthenogenesis in Starfish Eggs and the Condition of its Action....... 375

Tuos. H. Monrcomery, JR.
The Sex Ratio and Cocooning Habit of an Aranead and the Genesis of
Sex Rationse With aewoll i punes mec canioe aii «6 Meee a Ae 429

No. 4—June, 1908
N. M. STEVENS

The Chromosomes in Diabrotica vittata, Diabrotica soror and Diabrotica
12-punctata. A Contribution to the Literature on Heterochromosomes
and Sex Determination. With Three Plates...................-. 453

Victor. E. EMMEL

The Experimenal Control of Asymmetry at Different Stages in the Devel-
opmentofithe lobsters.) ome eee Erte one 471

C. M. CuiLtp

Physiological Basis of Form-Regulation. With One Figure .......... 485
H. H. Newman
The Process of Heredity as Exhibited by the Development of Fundulus
Hybrids. With Five Plates and Sixteen Figures in the Text ..... 503

C. C. GUTHRIE

Further Results of Transplantation of Ovaries in Chickens. With Three
Bigures acct raer siete rapt esi cterers reels, ee ieiateaer eer eS eee 563

H. S. JENNINGS
Heredity, Variation and Evolution in Protozoa. With Twenty-two

Figures SiS ce hectare ease eerieiee tee ann. Oe eee 577

DETERMINATION OF SEX IN HYDATINA SENTA

DAVID DAY WHITNEY

Mie oIntro duction: cram c yet eeieveieree vein etctetede ol 1eCotelsfoisislatavers:= nvessioLole/eale/ouclerarearfourtaletere: tae e/e}selel I
TE Material and methods: «<..2 2.0 cccceec cence creases deeceneensce cee eeseeuseecteee 3)
III Influence of temperature 4
1 Maupas’ experiments......-.00-es cece cece ete cee e rere eee t eee sence r een ers ces 4

2 Author’s experiments.........6 02-6 e secre cere eee e teen eee ener teen e eee ees 5

a Temperature 20° to 22°C. .... 1... e cece eee eee eet e eee t tn t eens 5

b Temperature 25° to 29°C... 1. eee eee eee eee tee teen ete e ene ees 8

c Temperature 14° to 15°C... 2. eee eee eee teen eter e teen eens 9

IV The relative number of eggs which a male-laying female and a female-laying female produc: 10
I Temperature 20° to 22°C... 21. cece cece tenet eet eee e cent ence eens II

2 Temperature 24° to 29° C.... 2.222 see eee erent ee tence ete cnt e erect ees II

V_ Early production of male-laying females in a family of daughter-females...............--. 13
Villm lin men celo Mt Oo samc isrovarcitereisveleilalalelsvol-porelaye ofosshaxeseps¥elcVerebesepsltaretceseleieds laidtsiel-(o[olaloriaretiere 15
1 Temperature 20° to 22°C... 2... ee eee ee ee eee ete teen entree cent es eees 16

2 Temperature 14° to 15° C.... 6.00 eee eee eee cent eee eee ee tenet ee eee ees 18

3 Temperature 25° to 26° C.... 1... cece eee eter e eect eee erence tees 18

aVI Male and female'strains........0..20.0ccecccs cer ecce cece cece etsccestesteneceerersce 19

VIII Production of fertilized eggs... ... 0... ce cece cece ec eee cere eet t eee eet e teen eee ene 23
YR S we ary ate ea Yay set saket ole vole aisieke orelalsatcla = ehtetaret=te)sfoin in) tove:wielalete\oL-setehegesonel se eisieieielape=;nheyaks 25

I INTRODUCTION

On account of the supposed influence of external factors in
determining sex in Hydatina senta, this rotifer has attracted much
interest in recent years. As is well known Hydatina produces
three kinds of eggs, viz: (1) parthenogenetic eggs which develop
into females; (2) smaller parthenogenetic eggs which develop into
males; and (3) fertilized eggs which develop into females. Each
female produces only one of these three kinds of eggs. ‘Thus
three types of females may be distinguished, viz: (1) females
which produce females parthenogenetically, or female-laying
females, 2 2; (2) females which produce males parthenogeneti-
cally, or male-laying females, @ 9; and (3) the sexual females
that lay fertilized eggs.

Tur Journat or ExreriMENTAL ZOOLOGY, VOL. V, NO. f.

2 David Day Whitney

Both female-laying females and male-laying females can be
impregnated by males, but on the former, impregnation is sup-
posed to have no effect. If the male-laying females are impreg-
nated by the male in the first few hours after they leave the egg,
such females produce fertilized eggs instead of parthenogenetic
male eggs, thus showing that male-laying females can develop into
sexual females that lay fertilized eggs.

The female-laying female can produce a family of daughter-
females, some of which may lay female eggs and others may lay
male eggs.

With the view of finding out the ratio in which these two classes
of daughter-females are produced under various conditions I have
carried out the experiments to be described.

Maupas found that a temperature of 26° to 28° C. would pro-
duce as high as 95 per cent of male-laying females while a tem-
perature of about 14°C. would produce as low as 5 per cent of
male-laying females.

Nussbaum, on the contrary, came to the conclusion that nutri-
tion and not temperature is the sex controlling factor. He found
that by starving the young females for the first few hours after they
emerge from the egg they would produce a high percentage of
males, but if they were fed at the time they leave the egg they pro-
duce a high percentage of females.

Punnett has carried out a few experiments along the lines laid
down by Maupas and Nussbaum and finds that neither tempera-
ture nor nutrition is influential in determining the sex. He finds,
on the contrary, that there are definite “‘sex strains.”’ Some
strains produce 40 to 50 per cent of males, others produce a very
low percentage, 2 to 5 per cent, while others produce no males at
all, although reared through as many as seventy-two generations.

The greater part of the work of the present paper was planned
and begun in the spring of 1906, under the direction of Prof. T.
H. Morgan, before the results of Punnett were published. Not
knowing how to obtain proper food cultures the rotifers all died
in July and the continuation of the experiments was deferred until
October, 1906.

Determination of Sex in Hydatina senta 3

Il MATERIAL AND METHODS

In the latter part of April, 1906, Hydatina senta was discovered
in great numbers in a small pool on the Palisades of New Jersey
near Grantwood. The pool was fed by a little stream or ditch
which carried away the drainage from several cottages. “The
ditch was an extremely favorable place for the growth of Euglena
viridis which collected in large patches on the sides and bottom.
Immense numbers of Euglena floated down into the pool at the
end of the ditch and served as food for the rotifers which abounded
there in countless thousands. Sometimes as many as 150 to 250
individuals could be drawn up by a pipette in a few cc. of water.

About May 15 the pool dried up completely. The ditch still
contained water but no rotifers were found in it after May 20. At
this time there were innumerable larve of insects in the ditch and
perhaps they exterminated the rotifers by feeding upon them.

In all experiments each individual female was isolated in a
square or round watch glass which contained about 5 cc. of water
and fed with Euglena, other protozoa and bacteria.

In order to obtain the Euglena and other protozoa a culture of
horse manure and water (one to two ounces to a quart) was made,
inoculated with Euglena and allowed to stand for two to three
weeks at room temperature. At the end of this time the green
coating of alg, Euglena, etc., could be removed from the sides
of the glass jar and served as an excellent food for the rotifers.

Great care was taken to keep these food cultures uncontami-
nated by rotifers. All watch glasses were placed in hot water
after each experiment in order to destroy all eggs which adhered
to the sides, thus preventing contamination of the following experi-
ments by eggs of the preceding ones.

The experiments at temperature of 24° to 29° C. were con-
ducted in an incubator. ‘Those at a temperature of 20° to 22° C.
were conducted on the laboratory tables at room temperature,
while those at a temperature of 14° to 15° C. were carried on in an
ice chest.

These rotifers are exceedingly hardy and can be very easily kept
in the laboratory throughout the year. In Mayof 1906 a Euglena
culture was prepared in a glass jar containing 2000 cc. of water

4 David Day Whitney

and a few rotifers put into it. The jar was covered so as to pre-
vent evaporation of the water. Rotifers have lived in it to this
time, April, 1907, although no more food material has ever been
added. It is absolutely necessary that the surface of the water be
free from a scum for the rotifers will die within a few hours if it
is present. It is safer, in order to keep the surface free, to tie the
horse manure in a muslin cloth and place it in a well covered jar
nearly filled with water.

III INFLUENCE OF TEMPERATURE

I Maupas’ Ex periments

The experiments of Maupas were so briefly described that it is
very difhcult to understand clearly how he obtained his results.

Nussbaum and Punnett are inclined to believe he determined
that a female was a male-laying or a female-laying individual
by the size of the eggs that she produced. Small eggs being
assumed always to give rise to males while larger eggs give rise to
females. Nussbaum has measured a series of both male and
female eggs and found that in some instances the two kinds of
eggs over-lap in size. ‘Thus he points out an error through which
Maupas’ results might have been obtained.

Isolating and counting the eggs of this rotifer would be exceed-
ingly tedious and require almost constant attention. As the sexes
can be readily distinguished at any period and as it requires only
36 to 48 hours for a female to mature and produce eggs it seems
to me extremely probable that Maupas must have allowed some
at least of the eggs to hatch before recording his results.

As his experiments are so few and briefly described it may be
well to present them here in order that they may be compared
with and interpreted by my own. Experiment I. Lot A, tempera-
ture 26° to 28°C. Five female-laying female sisters produced 104
eggs; 97 per cent developed into male-laying females. Lot B,
temperature 14°C. Five other female-laying females, which
were sisters of lot A, produced 260 eggs; 5 per cent developed into
male-laying females.

Determination of Sex in Hydatina senta 5

Experiment II, temperature 14° C. Five female-laying females,
kept from the time of hatching at this temperature, produced 110
eges; 24 per cent developed into male-laying females.

The same five female-laying females were then placed at a tem-
perature of 26° to 28° C. and produced 81 per cent male-laying
females.

Experiment III, temperature 14° C. Six female-laying females
which had been kept at this temperature from the time of hatch-
ing produced 34 eggs, of which 12 per cent developed into male-
laying females.

The same six female-laying females were then placed at a tem-
perature of 26° to 28° C. and allowed to produce 44 eggs, of which
95 per cent developed into male-laying females. These six females
were alternately placed at 14° C. and 28° C. several times and
always gave a high percentage of male-laying females at the
higher temperature.

2 Author's Experiments
a ‘Temperature 20° to 22°C.

Experiment I, October 24, 1906. A female-laying female was
isolated from a jar which was stocked with rotifers collected Octo-
ber 2, from the same pool in which the animals were found in the
preceding spring.

This strain was carried through twelve generations and the
percentage of male-laying females determined. Each female was
supplied with an abundance of food from the time of hatching,
isolated in a separate watch-glass, and kept upon the laboratory
table at room-temperature.

Table I gives the ratio of the mother individuals producing
male and female offspring in the 3264 daughter-females of 95
female-laying females in the twelve generations.

This experiment was made in order to obtain the percentage
of male-laying females produced at room temperature of 20° to
22° C., in order to be able to have some standard percentage of
male-laying females with which to compare the results of the
experiments conducted at lower and higher temperatures.

6 David Day Whitney

TABLE I
Record of the production of male-laying and female-laying females among the 3264 daughter-
females of 95 female-laying mothers.

Temperature 20° to 22° C.

No. | Eggs | Offspring | Per | | No. Eges | Offspring | Per
Gen. | 22 ale [> ae || cent -||Gen--) 299 oc | po ercireeceneenn ECCOE
| mother fe | oe yc Wh ears: mother laid (sional) Louse eine)
SS | eta | Se ape = see
| | | |
I I ar | ets Vere) | 48a 3 32 8 | 24 25
| | 4 15 3 12 20
I I 28. || 0: |) 28 ° | Ce Ne 223 Sie ess 18+
} 2 | 53 | 8 | 45 | ase | 6 48: 9) rte! Rage eae
| 3 27) hn: 23.«| «14+ | 7 25 o | 25 ro)
| | | 8.938 | 15) 23 tao
It I 440 cay | a7 38+ | 9 17 La be 23+
| 2 | 20.0) ara 325) 20h 10 48 15 3300 Stats
| | | II 48 | 16 33+
IV Te al ees I so | i+ | 12 43 4 39 | 9+
2 | 47 15 32 | Bit 13 44 | 10 34. |) 722-5
30 N52 elo Mh iea6 tao 14 49 | +14 35. | 28+
lisa asoonie 71/8 28 ulezo | Sx¢/ 1 Wasa ees 27 40
| 16 16 | 6 10 37+
My I 47 Our 47 ot 17 45 | 4 41 8+
2 44 2 42 Piaoe Ml 18 50 7 43 14
3 41 40 2+ | 19 | cpa il) A 28 12+
4 45 ° 45 | | 20 21) |" 0 21 °
21) 0) 2) See 17 19+
NAD pag ae Mie 12 20 27+ 2220 2 18 10
i) ee 45 4 41 8+ 230+) «925 ° 25 °
Saas o | 36 | © | |
eae illsa7 | go. | 14+ ar 49 8 40 16+
5 | 48 22 26 «| 45+ } | |
| XII I 38 3 35 7+
VIE]; 1 30 15 15 | ‘50 2 45 9 36 | 20
| 2 35 I 34 2+ 3 48 4 44 8+
3 | 27 ° 27 ° 4 31 5 26 16+
4 24 ° 24 ° 5 shee (aie) 26 | 33+
5 19 5 14 | 26+ | 6 31 10 21 32+
| latZ 54 To 440) 18+
Vill I 41 16 25 39+ es 45 6 39 13+
Panes 17 I 16 | s+ | 9 35 II 24 31+
3 47 18 29 38+ | 10 43 5 B85 u) Erte
4 26 ° ye | xr 38 3 35 iia
5 38 | ° 38 ° 12 27 9 18 33+
13 42 16 | 26 |- 38+
Ix I 24 | I 23 4+ 14 34 ei Og Bae
15 83 15 | 28 | 34+
x I 49 | 13 36 0 | 26+ | 16 37 6 | 31 16
axe 45 10 35 224+ | | 47 31 26 16+

Determination of Sex in Hydatina senta

TABLE I—Continued

Temperature 20° to 22° C.

Now Eeeeet| a Obspaugs |) be Noo ees ||) Otspung, || Se
Gen. Se a TERE. || |i ea | ass Gen. 2h) heat Ise —
mother | au oon] Lo) {0} ae mother Gs ae? 29
XII 18 26 7 19 | 22+ || XII | 30 16 5 II
19 33s S| 3r 9 oe A
20 4 | 3 41 6+ 32 18 8 | 10
21 20 ° 20 Qi | 33 20 6 14
Bam Aleetsc i ltee 36 | 20 | 34 47 14 33
| |
} 31 ut ZO E350) 35 x9) 4 15
24 28 10 i | 35+ 36 26 8 18
25 15 3 2 [20 | 37 II ° II
26 34 I 33 2+ 38 37 II 26
27 15 ° 152) Ce sl 3934 19 15
28 14 3 II 20 40 25 6 19
29 6 I 5 | 16+ | 41 35 4 31

|
\
1

cent

31+
Dict

44+

3°

The nature of the sex-producing power of the daughter-females
of each individual mother is given separately in order to show that
the ratio between the daughter-females producing male and female
offspring varies with different mother-individuals, and also varies
as much in the daughter females of sister-mother-individuals.

Summary of each generation in Table I and also the final summary of all the generations taken

mother

TABLE II
together.
Temperature 20° to 21° C.
Offspri Per
Werceonl EE eesa | les aR:
Sb \inoeher laid 29 29 Sad Gen.
Tye 31 15 16 | 48+ | VIZ |

I 3 108 12 96 i+ | VOI
Ii 2 go 31 59 34+ ee
x

IV 4 185 39 146 21+
XI

Vv 4 177 3 174 I+
xi

VAS WG 208 45 163 21+

Eggs Offspring Per
aid Ee | ae cent
Sa a?
_ | a al
135 21) 114 | 15+
|
|
169 35 | 134 | 20+
24 I |} 23 4+
819 176 643 -| 21+
49 8 41 16+
1269 268 | roor | 21+
3264 654 | 2610 | 204

8 David Day Whitney

Table II gives the summary of each generation and the final
summary of the twelve generations.

In this experiment 95 mother-individuals produced 3264 daugh-
ter-females of which 20+ per cent were male-laying females.

It will also be noted that the percentage of male-laying females
varied in the different generations from I + per cent to 48+ per
cent regardless of the number of isolations in each generation.

b Temperature 25° to 29° C.

Experiment II, October 26. Two female-laying females were

TABLE III

Record of the production of male-laying and female-laying females among the 208 daughter-females
of 26 female-laying mothers.

Temperature 25° to 26° C. Temperature 26° to 29° C.
| No. Leges| Ofspring| Per No. Eggs Offspring | Per
Gen.| 99 | ale __| cent Gen.| 29 laid | — | cent
| mother | ] 82/29} oe | mother J 2)9 9 | oS?
| | | | | | |
StrainIV) I) 1 | 17| o| 17] © Strain TT IX | I | Werey [Naz | °

| | 2 || go" | ral °
|

I I (Ts ae Ly | we (or | | | |
| | xX 1 |e 31, ONS
StrainIII| I I 17 corals 4 | o | 2 Sa sol) 5 °
| {<3} 1h Srh3|) an Fo) IE100
Ul I 13} 4] 9] jot+ AL || <t\feo: | ex °
5 8| 2 | 6 | 24+
peel I 16 | 2/14] 12+ 6 6) t] 5 | 16+
| oh |W 2 | 6| 24+
Vil | I 16 | Mal 12+ 8 oat foie} °
| | 2 | 10 | 3 7 30 9 | 5 2 | 3) 4°
3 TE | (6:5 Gi Sat Tor | 143) 225i) 450
| 4 25 | 13 | 12 | 52+ are Nie CNAs aac
: 725 |/91\) 05203 ae
13 i at I Ce 0c
| 1 | 10 3| 7] 30
| ef | © | 100
| 26/208 46 |162 | 22+

isolated from the same stock jar in a similar manner as in Experi-
ment I, and placed in an incubator. ‘Two generations from one

Determination of Sex in Hydatina senta 9

individual, strain [V, and six generations from the other individual,
strain II],were recorded. Six generations were kept at a tempera-
ture of 25° to 26° C. and two generations at 26° to 29° C.

Table III gives the results of the experiment obtained at this
higher temperature. The 26 female-laying mothers from eight
generations, and from two strains, produced 208 daughter-females
of which 22 + per cent were male-laying females. ‘This percent-
age is practically the same as that obtained at room temperature.

@ Wemperature 14° to 15°C.

Experiment III, October 8. A female-laying female was isolated
from stock jar as in Experiment I, and placed in an ice chest. A
record of only a few of her offspring was kept and is shown in

Table IV.

TABLE IV

Record of the production of male-laying and female-laying females among the 167 daughter-females
of 7 female-laying mothers.
Temperature 14° to 15° C.

No. of |

eo | Eggs ; Ofspring ; Per cent
mother | laid | Fed oo | 99 Fe
I 48 47 11 36 23+
2 so «| «28 4 24 14+
3 30 20 3 17 15
4 | 22 9 2 7 22-+-
5 41 19 5 14 26+
6 | 36 15 ° 15 °
7 | 51 2 ro) | 19 34+
7 | 278 167 35 132 20+

Out of 167 daughter-females from 7 different mothers 20+
per cent were male-laying females. “Uhe mother-individuals were
reared at this low temperature as well as the daughter-females.

The percentage of male-laying females is about the same as
that obtained at temperatures 20° to 22° C. and 25° to 29° C.

The foregoing results agree with those obtained by Nussbaum
and Punnett but seem contrary to Maupas’ results.

10 David Day Whitney

IV THE RELATIVE NUMBER OF EGGS WHICH A MALE-LAYING
FEMALE AND A FEMALE-LAYING FEMALE PRODUCE

It seems evident from Maupas’ account of his own experiments
that he did not isolate each female-laying mother and each one
of her daughter-females but kept the female-laying mothers to-
gether in one dish and their daughter-females together in another
dish.

If it is assumed that Maupas made no mistake in determining
the sex character of the eggs before they hatched, or even that he
allowed all eggs to hatch before he recorded their sex character,
his results can be easily explained.

TABLE V

The number of eggs laid by each of 13 sisters, of which 6 were male-laying and 7 were female-laying,
showing that the average number of eggs laid by each of the two kinds of females is very nearly the
same.

Temperature 20° to 22° C.

I 46 I 47
I 50 I 38
I 47 Li 37
I 46 I 43
I 41 I 38
I 31 I 45
I 38

6 261 434 7 268 408 -

He gives no results of experiments conducted at a tempera-
ture midway between 14° and 28° C. but only results obtained at
these two extremes. The results that were obtained at 14° C.
may very likely be identical with those that could have been
obtained at a room temperature around 20° C,

Maupas recognized the fact that male-laying females produce
eggs faster than female-laying females but makes no mention of
the number of eggs that each kind of female may produce at dif-
ferent temperatures. He seems to assume that they always pro-

Determination of Sex in Hydatina senta II

duce about an equal number, 40 to 50 each, but the following
experiments will show the error of this assumption.

I Temperature 20° to 22° C.

Experiment IV, November 5. Of 13 sister individuals kept
at room temperature and with the same amount of food 6 pro-
duced male eggs and 7 produced female eggs. ‘The average num-
ber of eggs produced by each female was nearly the same. ‘The
results are shown in Table V.

2 Temperature 24° to 29° C.

Experiment V, November 9. ‘Three lots of sister-individuals
from three different mother-individuals were kept in an incubator

TABLE VI
Record of the number of eggs laid by 11 sisters, of which 6 were male-laying and 5 were female-laying,
showing that the average number of eggs produced by the male-laying females is about two times as
great as the average number produced by the female-laying females.

Temperature 24° to 25° C.

II sisters

7
A =) route) 9
a? | 2 2
|

oO z 2

| Av.

Mother Eggs we Mother | Eggs | if

= —- —_ ls =
I 37 I 16
u 35 1 17
I 26 I 16
I 38 I II
I 26 I 10

I 22
e 184 305 se a ze 14

and the number and sex character of the eggs that each produced
was very carefully noted. The results are shown in Tables VI,
VII and VIII. The records of the individuals in Tables VI and
VII were taken at the same temperature of 24° to 25° C. while
those of Table VIII were taken at a higher temperature of 26°
to.29° C.

These tables show a decided change in the ratio between the
number of eggs produced by a male-laying female and a female-
laying female. As the temperature is raised the female-laying

12 David Day Whitney

TABLE VII

Record of the number of eggs laid by 21 sisters, of which 9 were male-laying and 12 were female-
laying, showing the average number of eggs produced by the male-laying females is about two times
as great as the average number produced by the female-laying females.

Temperature 24° to 25°C.

21 sisters
rr Peles se =
oe oe | 29 g
Mother | Eggs aN | Mother Eggs Ny
loa fe ee
I 25 «| I 6
I 39 | ee 14
I 34 I 16
I 37 I II
I 28 | | I 10
I 25] | I 12
I 29 | st 12
I 23504 | I 9
I 22. 4 | I 17
\fesjeat 14
|
| I 16
A Pee se | a | F ae ;
9 261 294 lh 33 158 13
TABLE VUI

Record of the number of eggs laid by 14 sisters, of which 2 were male-laying and 12 were female-lay-
ing, showing that the average number of eggs produced by the male-laying females is nearly four
times as great as the average number produced by the female-laying females.

Temperature 26° to 29° C.

14 sisters
a5 Se a ey
oun) a | ; | eye} ie)
Mother | Eggs ox Mother | Eggs ENG
| = = —
| |
I 23 | I 3
| ee | 1 5
| | I I
| I 8
I 6
I 8
: 3
| a 5
| 1 4
| I 4
| : Dial
Al od ees oe feces
2 | 42 [he oan | 12 66 Ci 53

Determination of Sex in Hydatina senta 13

females produce fewer and fewer eggs while the decrease in the
number of eggs produced by male-laying females is not as great.
Table LX shows a rough approximation of the ratio in which the
male and female eggs are produced at these different tempera-
tures.
TABLE IX

The approximate ratios in which the males and females are produced at different temperatures,
as seen in Tables V-VIII.

1 | x | Temp. 20°to22°C. Table V
2 1 Temp.24°to25°C. Table VI-VII
4 1 Temp. 26°to 29°C. Table VIII

From the foregoing experiments and tables it is evident that
temperature has nothing to do directly with determining sex in
Hydatina senta but indirectly it determines the number of each
sex produced by regulating the number of eggs that each kind of
female lays. At a temperature of 20° to 22° C. the male-laying
and female-laying females lay about the same number of eggs
each, but at a higher temperature of 26° to 29° C. the male-laying
females lay about four times as many as the female-laying females.

V EARLY PRODUCTION OF MALE-LAYING FEMALES IN A FAMILY OF
DAUGHTER-FEMALES

None of the previous workers with Hydatina senta have iso-
lated the eggs of a female-laying female in the order in which they
were produced to determine whether there is any tendency for
the earlier laid eggs to produce more male-laying females than the
later laid eggs.

In my experiments in which all the daughter-females of each
individual mother were carefully isolated and the sex character of
their immediate offspring was recorded it is clearly shown that
the male-laying daughter females appear among the earlier ones
in the family rather than among the later ones.

In Diagram 1 the plotted line indicates the production of male-
laying females among the 472 daughter-females of eleven mother-

14 David Day Whitney

individuals, each one of which produced 40 to 44 (average 421%

eggs. ‘The eggs were allowed to hatch in the dish with each
mother and the young daughter-females isolated soon after hatch-
ing. Their different sizes would indicate their relative ages and
thus the approximate order in which the eggs were produced.
The young daughter-females were isolated in lots from 1 to 8.
This manner of isolation is subject to some error but on the whole
gives a fairly good approximation of the truth.

Pe Pee
[ea]
CEE PEEEEHE
a ae
y N HH fs
ran Reece poet

ducing male-laying females

Number and location of eggs pro-

NUMBER AND ORDER OF FEMALE EGGS PRODUCED

Diagram 1 Record of the egg production of 11 female-laying females from Table I, showing in
which part of the egg laying period the male-laying females were produced. Each female laid 40 to
44 (average 42+) eggs. Of the 472 daughter-females 20+ per cent were male-laying females.

Nearly all of the male-laying females were produced among the
first 28 eggs laid. Only two male-laying females were produced
from the twenty-ninth to the forty-second laid eggs. Of the
daughter-females 20 + per cent were male-laying females.

Diagram 2. ‘This is to show the same point as Diagram 1.

oping into male-laying females

Number and location of eggs deve!-

NUMEER AND ORDER OF FEMALE EGGS PRODUCED

Diagram 2 Record of the egg production of 12 female-laying females from Table I, showing in
which part of the egg laying period the male-laying females were produced. Each female laid 35 to 39
(average 36+) eggs. Of the 441 daughter-females 16+ per cent were male-laying.

Determination of Sex in Hydatina senta 15

Twelve other female-laying females, each of which laid 35 to 39
(average 36+) eggs, produced all their male-laying daughter-
females among the first twenty-four eggs laid. This is a clearer
case than Diagram 1, because there are no scattering male-laying
females among the later produced eggs. Of the daughter-females
16+ per cent were male-laying females.

In these two diagrams the mother-individuals were not specially
selected but the record of all mothers, in Table I, producing 40
to 44 daughter-females, is shown in one diagram and the record of
all mothers, in Table I, producing 35 to 39 daughter-females is
shown in the other diagram. The numbers 35 to 39 and 40 to 44
were chosen because they seemed more likely to be the normal
than a higher one.

These results, together with those obtained at different tem-
peratures throw a great deal of light upon Maupas’ results. In
-his experiments the highest percentages of males was always
obtained from mothers which developed from early laid eggs.

In Table I it is seen that an individual mother may produce
O to 40+ per cent of daughter male-laying females. This fact
must also be taken into account when explaining Maupas’ few
experiments. ”

VI INFLUENCE OF FOOD

Nussbaum supported Maupas’ conclusions that external factors
can change the sex ratio in Hydatina but explains this change as
being due to poor nutrition of the females and not due directly
to the influence of temperature. At the higher temperature the
processes of metabolism are taking place so rapidly that the ani-
mals cannot eat food and assimilate it fast enough to prevent their
tissues from being in a semi-starved condition. Nussbaum’s
experiments seemed to show evidence that young females which
are starved for several hours as soon as they leave the egg produce
a higher percentage of males than those that are fed from the
moment they hatch.

In many of his experiments he kept many individual females
together and did not follow the history of each individual sepa-
rately.

16 David Day Whitney

Punnett has pointed out that all the starved females of Nuss-
baum’s experiments did not produce males, which invalidates his
general conclusions.

Punnett has isolated female eggsof a “pure female strain,” and
after they hatched starved the young females from 2 to 20 hours
but no males ever appeared, although the young females were
starved for several consecutive generations.

I have followed the history of many females which have been
starved for several hours immediately after hatching at a tempera-
ture ranging from 14° to 29° C. and have found no trace of
evidence that a higher percentage of male-laying females is pro-

duced.

I Temperature 20° to 22°C.

Experiment I. Sixty-two eggs were selected at random from
the sets of eggs produced by four female-laying females. They

TABLE X TABLE XI
Record of the sex character of the eggs laid Record of the sex character of the eggs laid
by 27 sisters, 15 of which were without food for by 45 sisters, 11 of which were without food for
the first 6 to 26 hours after hatching and 12 the first 21 to 26 hours after hatching and 34
were abundantly supplied with food from the were abundantly supplied with food from the
moment they hatched. moment they hatched. ~
Temperature 20 to 22° C. Temperature 20 to 22° C.

ee a | hours -
% 2 : II 21-26 | 2
; aS 2 13 fed rol
5 a i 21 fed | 9
I 12 2 a La eS
I 16 Q
2 21 g
I 23 rol
I 2 fof
2 fed rol

were placed in “Great Bear” spring water, such as is sold in New

York City for drinking purposes, and allowed to hatch. After

Determination of Sex in H ydatina senta 17

hatching each daughter-female was kept in this water without
food from 6 to 71 hours.
Of tog daughter-females from the same mother as the above,

62 were well supplied with food from the moment they hatched.
Tables X to XIII give the detailed results and Table XIV gives
the summary. The mother-individuals of Tables X and XII

TABLE XII TABLE XIII

Record of the sex character of the eggs laid enoud Cees Sea of Rea
5 we 3 by 46 sisters, 5 of which were without food for

by 53 sisters, 31 of which were without food for Fee Beek:

the first 11 to 59 hours after hatching and 22 Gre Te So 0) 7 Ce : ey Aa ng One 2

eereepundantly; supplied wwithfoad) orl the were abundantly supplied with food from the

mom hey hatched.
moment they hatched. oment they hatche

Temperature 20 to 22° C. Temperature 20° to 22° C.

| Character of eggs Character of eggs

Sister- | Starved from Sister- | Starved from

were sisters. The 11 sisters-individuals of Table XI were
starved in filtered boiled spring water placed in sterilized test tubes
with cotton stoppers.

The percentage of male-laying females among all the starved
daughter-females is slightly lower than that of those which were

fed.

18 David Day Whitney

TABLE XIV

Summary of Tables X to XTIT, showing the percentage of male-laying females that occurred among
the females which were without food for the first 6 to 71 hours after hatching, and also the percentage
of male-laying females which occurred among the females that were abundantly supplied with food from

the moment they hatched.
Temperatnre 20° to 22° C,

Starved Character of eggs |

Indi- | from produced Per cent
viduals! time of | @
hatching fou 4
hours
62 | 6-71 12 50 19+
1og | fed 27 82 | 24+
171 | 39 132 | 22+

2 Temperature 14° to 15° C.

Experiment II, October 29. “Two female-laying females were
reared at this temperature and their daughter-females isolated.
Thirty-five daughter-females were without food from 11 to 64
hours after they left the egg, and 49 were abundantly supplied
with food as soon as they hatched.

The detailed results are shown in Tables XV and XVI while
Table XVII gives the summary.

The difference between the percentage of males produced by
those starved and those fed is not very great and probably means
nothing.

3 Temperature 25° to 26° C.

Experiments III, October 31. “Twenty-six female eggs from
several individuals were produced at this temperature and as soon
as they hatched the young females were starved from I to 13
hours. 19+ per cent of these starved females produced male
eggs.

Tables XVIII and XIX give the detailed history and Table XX

gives the summary.

Determination of Sex in Hydatina senta 19

These three experiments, including Tables X to XX, clearly dem-
onstrate that food has no influence in determining whether a
female shall produce male or female offspring.

TABLE XV TABLE XVI
Record of the sex character of the eggs laid Record of the sex character of the eggs laid
by 49 sisters, 19 of which were without food for by 35 sisters, 16 of which were without food for
the first 11 to 41 hours after hatching and 30 the first 20 to 64 hours after hatching and 19
weie abundantly supplied with food from the were abundantly supplied with food from the
moment they hatched. moment they hatched.
Temperature 14° to 15° C. Temperature 14° to 15° C.
Sister- | Starved from Sa CeCNICEES Sister- | Starved from eeoerec cau Ee
individ- | time of hatch- __Produced individ- | time of hatch- |__ Produced
uals ing 3 ro) uals ing 3 9
| |
hours hours |
4 II g 2 20 9
I 20 te) I 21 cr ||
2 | 20 fot 2 21 co]
3 21 2 2 23 | @
I 23 9 I 46 fol
5 25 g I 46 }
I 25 fol I 48
I 27 9 1 53 |
I 41 ge I 53 lees,
4 fed rot I 58 2
26 fed 9 I 61 of
v — ae ce I 61 g
I 64 fot
5 fed fot
14 fed g

VII MALE AND FEMALE STRAINS

Punnett says: “My experiments have led me to the conclusion
that among the rotifers I used were certainly three different types
of thelytokous (female-laying) females, viz:

A Females producing a high percentage of arrenotokous
(male-laying) females.

B Females producing a low percentage of arrenotokous
females.

C Purely thelytokous females producing no arrenotokous
females”’ (p. 226).

20 David Day Whitney

TABLE XVII

Summary of Tables XV to XVI, showing the
percentage of male-laying femal s that occurred
among the females which were without food for
the first 11 to 64 hours after hatching, and also
the percentage of male-laying females which
occurred among the females that were abun-
dantly supplied with food from the moment they
hatched.

Temperature 14° to 15° C.

Starved Character of eggs |

i- | Per
aac | from produced

vidu- | time of sae | oe

als hatching a | 2 Ge

35 | 11-64 8 27 22+

49 fed 9 40 18 +

84 H7 |||| 167 20 +

TABLE XIX

Record of the sex character of the eggs laid
by 19 individuals females which were without
food for the first 1 to 13 hours after hatching.

Temperature 25° to 26° C.

| | Character of eggs

re Starved from
Individ- time of hatch- produc’) =
uals :
ing rol fo}
hours
I I | 2
2 2 | Q
I 3 | ice
3 3 %
I 4 fos
I | 5 | of
2 5 2
2 6 | 2
I 8 fof I
3 10 | &
2 13 | 2

TABLE XVIII

Record of the sex character of the eggs laid
by 15 sisters, 7 of which were without food for
the first 7 to 13 hours after hatching and 8 were
abundantly supplied with food from the moment
they hatched.

Temperature 25° to 26° C.

| Starved from | Character of eggs

suis m- | time of hatch- produced
dividuals :
ing fot 9
hours |
2 7 | 2
2 10 ee
: 10 a |
2 13 | 2
I fed rol |
7 fed °
TABLE XX

Summary of Tables XVIII to XIX, show-
ing the percentage of male-laying females that
occurred among the 26 females that were with-
out food for the first 1 to 13 hours after hatch-
ing.

Temperature 25° to 26° C.

Determination of Sex in H ydatina senta ZT

Punnett realized that these conclusions were based on rather
scanty data. His data can be shown to be entirely insufhcient.
His type 4 is based upon only one experiment which extended
through 23 generations and included 109 individuals. 42 + per
cent of these 109 individuals were male-laying females.

Type C is based upon much more evidence, but it is not sufh-
cient to warrant a decisive conclusion.

In October, 1906, I started a strain or pedigree culture which
extended through 62 generations including 167 mother female-
laying females and 3959 daughter-females. ‘This strain was kept
at room temperature of 20° to 22° C. Its history is recorded in
Tables I and XXI.

Table I. Out of 3264 daughter-females from g5 mother-indi-
viduals which extended through 12 generations 20+ per cent
were male-laying females.

Table XXI. In r5 generations, XIII to XXVIII, including 76
daughter-females from 15 mother-individuals only 9 + per cent
were male-laying females.

In 17 generations, XXIX to XLV, including 208 daughter-
females from 17 mother-individuals no male-laying females ap-
peared. Ingeneration XLVI the first 327 daughter-females from 18
mother-individuals yielded 48+ per cent of male-laying females.

The next 11 generations XLVII to LVII including 58 daughter-
females from 11 mother-individuals gave 29 + per cent male-
laying females.

These results show that a strain producing a higher percentage
of male-laying females can develop into a strain yielding a much
lower percentage, or even into a strain yielding no male-laying
females at all. Furthermore, the apparently pure female-laying
female strain can develop into one which will give a very high
percentage of male-laying females.

Thus the three strains or types of Punnett can be found in one
strain and each is capable of giving rise to the other types accord-
ing as the data is scanty or extensive.

The high percentage of male-laying females in generation XLVI
can be readily explained by the results shown in DE seme I and 2
which clearly demonstrate that the male-laying eles are pro-

22 David Day Whitney

duced earlier in a set of eggs than are the majority of the female-
laying females. In this generation the 18 mothers produced
only an average of 18 + eggs each, because the experiment was
discontinued at this point.

TABLE XXI

Continuation of the strain of which the beginning is recorded in the 12 generations of Table I.

ay Daugh- 29 | Daugh-
Gen. maopkee |. ter 2 ae 2 9 Gen. Mother | _ ter 9 32 99

isolated | isolated
XIII I 6 I 5 XXXVUI Tae hy 37, ° 37
XIV I Gye ar 5 XXXIX I | 6 ° 6
RV) ia Parla as x! Mere a6 o | 26
XVI I 6 ° 6 XLI Ta 25 ° 25
XVII I 2 ° 2 XLII Ti] 14 ° 14
XVIII I I ° I XLill rife 78 ° 18
xIX I 5 ° 5 XLIV I 17 ° 17
xx I 6 | ° 6 XLV I 18 ° 18
XxI I 6 ° 6 XLVI 18 327 160 167
XXII I 6 | 3 3 XLVII I I ° I
xxmm| 1 Pant ee al XLVI] 1 6 al 2
XXIV I 6 ° 6 XLIX I 6 3 3
XXV I 6 ° 6 L Teal 6 2 4
XXVI I 6 I 5 LI I 6 ° 6
XXVII I I ° I LIL I 6 ° 6
XXVIII I 2 | 1 I LUI I | 6 ° 6
XXTX I Se le 30 5 LIV I 6 5 I
XXX I 6 ° 6 LV 1) 5 2 2}
XXXI I 4 ° 4 LVI I | 6 5
XXXII I I ° I LVI I 4 ° 4
XXXII I 5 ° 5 LVI 3 6 ° 6
XXXIV 1 Gia sl xe 6 LIX 1 6 ° 6
xXXxXXV I 6 ° 6 LV 2 6 ° 6
XXXVI I 6 ° 6 LXI 6 ° 6
XXXVII I 6 | o 6 LXII 2 4 I 3

How a seemingly pure female-laying female strain is obtained
when only a few individuals are isolated from each generation
while a parallel strain does not yield the same results is not yet
clear. It may be due to some trick of selection in isolating the
young females of each generation.

Determination of Sex in Hydatina senta 23

If a large number, 45+ per cent, of the daughter-females of
one mother produce males it does not necessarily follow that a
high or a low percentage of the daughter-females of the next gen-
eration will produce males. Nor does it seem to be true that if
the daughter-females of one generation produce all female off-
spring that the daughter-females of the following generation will
do so. ‘Table XXII shows the history of both classes of daughter-
females in five generations.

TABLE XXII

Record of all the female-laying females that were isolated in five consecutive generations, showing
that there is no constant relationship between the percentage of male-laying females that are produced
by a mother and the percentage of male-laying females that are produced by the daughter-female in
the next generation.

Off- Moth- Off- Off- Moth Off-
Gen. Sisters spring er spring Gen. | Sisters spring | er | spring
a2 | 99) 99 | ae |ee|ee
I I AV 5 15 15
II 16 ] 36 I 34
ic ° 27
Ut 4 ° 45 | ° 24
2 42 | § 14
I 42
i) | 47
it
IV 5 12 20
4 41
° 36
7 40
22 | 26

VIII THE PRODUCTION OF FERTILIZED EGGS

The winter or fertilized egg is supposed to be the male partheno-
genetic egg which has been fertilized. This produces a female.
The egg has much more yolk material and a thicker shell than the
male parthenogenetic egg. A female produces from twelve to
twenty fertilized eggs, while a male-laying female produces from
forty to fifty parthenogenetic eggs. In order to obtain fertilized
eggs males must copulate with very young male-laying females.

24 ; David Day Whitney

In a few experiments, comprising several hundred females which
had copulated with males when very young, Maupas found that the
percentage of females producing fertilized eggs was the same as
the percentage of male-laying females from several hundred fe-
males that never had copulated with males. He concluded that
the fertilized egg is the male parthenogenetic egg which has been
fertilized.

I have repeated his experiments on a smaller scale and the same
results were obtained. Furthermore, the producers of fertilized
eggs appeared among the early laid eggs of the mother-individuals.

Diagram 3 shows the occurrence of the layers of fertilized eggs
among their sister-individuals from five mothers which _pro-
duced 125 eggs.

Number and location
of eggs developing into
layers of fertilized eggs

NUMBER AND ORDER OF FEMALE EGGS PRODUCED

Diagram 3 Record of the egg production of five female-laying females showing in which part of
the egg-laying period the layers of fertilized or ‘‘winter eggs” were produced. Each female laid 21
to 27 (average 25) eggs. Of the 125 daughter-females 36 + per cent laid fertilized eggs.

Many males, fifteen to twenty, were constantly kept in the
dishes with each of the five mothers, so that as each daughter-
female emerged from the egg there were many males present.

Only one male-laying female appeared among the 125 daughter-
females. ‘This experiment was conducted in the fourteenth gen-
eration of the strain in Tables I and XXIJ, soon after the isolation
of the large numbers in generation XII which gave 21 + per cent
of male-laying females.

Of the 125 daughter-females 36 + per cent produced fertilized
eggs. This percentage is high because the mother-females pro-
duced, on an average, only twenty-five eggs each, but if they had
produced forty eggs each the percentage would have fallen to

Determination of Sex in Hydatina Senta 25

about twenty-three, provided that there had been no more mothers
of fertilized eggs to have been produced. “Vhe Diagram 3 shows
only one occurring between the eighteenth and twenty-seventh
egg.

The number and order of occurrence of the mothers of fertilized
eggs together with the number and occurrence of the layers of
male eggs in parallel sets of daughter-females seem to indicate
that the Sayer of male eggs and the layers of fertilized or winter
eggs are identical at one stage of their life.

In another species of rotifer, Asplancha, Lauterborn has observed
winter eggs and male embryos in the same individual. Among
the Daphnia, Issakowitsch has found that the same female may
produce winter eggs and male eggs.

Therefore it is not unreasonable to suppose that the immature
male-laying female of Hydatina senta is capable of developing
into a layer of fertilized eggs or a layer of male eggs, according
to the impregnation or lack of impregnation by the males

IX SUMMARY

1 Temperature has no influence in determining the sex of
Hydatina senta.

2 About 22 per cent of the females at any temperature from
14° to 29° C. are male-laying.

3 A male-laying female produces eggs faster than a female-
laying female and at a temperature of 25° to 29° C. a male-lay-
ing female produces more eggs throughout her lifetime than a
female-laying female.

4 The male-laying females occur in the early part of a family
of daughter-females.

5 Starving the young females for the first few hours after they
hatch does not cause them to produce a higher percentage of male
eggs.

6 ‘There are no strains that constantly produce a high or a
low percentage of male-laying females.

7 The “pure female-laying female strain” can give rise to the
normal percentage, 22 +, of male-laying females.

26 David Day Whitney

8 The male-laying female may produce fertilized or winter
eggs, provided that she has been impregnated by a male at the
proper time.

Zoological Laboratory
Columbia University

May 1, 1907

BIBLIOGRAPHY

Hupson, C. T., anpD Goss, P. H., °89—The Rotifera.
IssakowrrscH, A.—Geschlechtsbestimmende Ursachen bei den Daphniden.
Biol. Centralb., xxv, 1905.
LautTersorn.—Ueber die zyklische Fortpflanzung limnetischer Rotatorien. Biol.
Centralb., xviii, 1898.
LeussenN.—Contnbution a |’étude du developpement et de la maturation des ceufs
chez Hydatina senta. 4a cellule, xiv, 1808.
Maupas, M.—Sur la multiplication et la fécondation de |’Hydatina senta, Ehr.
C. R. AcuSe: ‘Paris; cxi, 1890.
Sur la fécondation de |’Hydatina senta, Ehr. C. R. Ac. Sc. Paris, exi,
1890.
Sur le déterminisme de la sexualité chez |’Hydatina senta, Ehr. C. R.
Ac. Sc. Paris, cxiii, 1891.
Nussspaum, M.—Die Entstehung des Geschlechts bei Hydatina senta. Archiv
fiir mikroskopische Anatomie, xlix, 1897.
Punnett, R. C.—Sex-determination in Hydatina, with some Remarks on Par-
thenogenesis. Proceedings of the Royal Society, B. vol. 78, 1906.

From the Havemeyer Chemical Laboratory, New York University, New York

A NEW EXPLANATION OF THE MECHANICS OF
MITOSIS

ARTHUR B. LAMB

Wirth Two Ficures

The almost universal recurrence of essentially the same regular
arrangement of the chromatin substance in dividing cells indicates
emphatically that the same very definitely acting force or complex
of forces is operative in them all. Numerous suggestions have
been made as to what this omnipresent force may be, but none of
them have been able to meet the many requirements of the prob-
lem. Moreover, our real knowledge of the whole matter is so
scanty that any explanation seems at present a little premature.
I am, nevertheless, going to offer still another explanation of this
phenomenon; first, because it may prove suggestive to others and
may prompt fresh observation, and second, because it calls attention
to a phenomenon which deserves the consideration of cytologists,
whether it has any application to the present case or not.

The marked polarity which mitotic figures exhibit, best de-
scribed by saying that they resemble the configuration assumed by
iron filings between unlike magnetic poles, together with the move-
ments which the chromatin substances execute about one another
oblige us to believe that this unknown force is of a polar nature,
that is, acts outward from a center and exerts its influence at a
distance. ‘The only objection to this conclusion is that a crossing
of astral rays has been observed. ‘The lines of force in the field
of any polar force cannot, however, cross, and consequently the
astral rays which would be assumed to follow these lines of force
also cannot, or should not, cross. This crossing, though certainly
real, is not, apparently, the prevailing condition, and it can be

THe Journar or Experimenta Zo6.ocy, VOL. v, No. I.

28 Arthur B. Lamb

explained on the assumption of an intermittent or non-synchronous
activity of the centers, as Reinke’ has shown.’

Assuming, then, the existence of some polar force exerting its
action at a distance, we are confronted with two possible alterna-
tives regarding the sign of this action. “That is, we may imagine
either that the centrosomes attract, or that they repel each other.
Wilson® has urged that the astral centers represent centers of trac-
tion, caused, perhaps, by a volume change at those places. This is
in entire agreement with the configuration assumed by the astral
rays and the spindle fibers. They simulate the magnetic field
between opposite poles, as pointed out above. But this view is
quite at variance with the actual movements of the centrosomes.
They move apart, even at a stage when astral rays are well devel-
oped and hence seem to repel each other and not attract as they
ought if they represent opposite poles. Lilliet adopts the other
alternative, as did Meves.° He considers the astral centers to
repel each other. In this way he explains the movements of the
centrosomes satisfactorily enough, but is confronted by the difh-
culty of accounting for the configuration of the astral rays. Lillie
assumes that electric charges located on the centrosomes are the
particular forces which produce the repulsion. He would explain
the unexpected configuration of the fibers and the astral rays by
the rather dubious assumption of a localized positive inter-astral
area which superposes its effect on the purely repellent action of
the astral centers. Looking at the matter more closely we see
that for every unit of negative electricity on the chromatin sub-
stance there should be a corresponding unit of positive electric-

1 Reinke, Fr.: Arch. f. Entwicklungsmech., ix, 1900.

*Rhumbler: Ibid., iii, iv and v, 1896, 1897 and 1899, has suggested a non-polar force to explain the
astral rays independently progressing rays of crystallization out of a supersaturated solution. While
avoiding the difficulty of crossed fibers this explanation encounters the still more formidable one of
accounting for the universal occurrence of curved fibers.

8 Tbid., xiii, p. 354-395, 1901. See also his book, The Cell in Development and Inheritance, 3d
Ed. The Macmillan Company, New York. It is a pleasure to express my thanks for a most profitable
discussion of this whole question with Professor Wilson, who, it seems, had already considered the pos-
sibility of a hydrodynamic explanation.

4 Amer. Jour. Physiol., xv, 46-84, 1905.

5 Ergebn. d. Anat. u. Entwick., vii, viii. Merkel u. Bonnet.

The Mechanics of Mitosis 29

ity in or on the surrounding aqueous solution. Moreover, since
this aqueous solution contains inorganic, ionized salts it must be a
conductor of electricity, and the positive charge must be distrib-
uted over the whole solution. Any localized positively charged
area in the electrolyte, except for the supposed “double layer”
around each charged particle seems, consequently, unlikely.
Lillie has encountered a similar difficulty in accounting for the
configuration of the chromosomes. ‘They ought not only to be
repelled from the astral centers but also to migrate toward the
boundary of the equatorial plate. This latter thing they do not do,
and Lillie is therefore again obliged to make the assumption of a
localized positive inter-astral region.

There is, however, another force which might well come into
play here, which so far as I know has not been mentioned in this
connection before, and which involves none of the objections
urged against the electrostatic explanation. I refer to the mutual
repulsions and attractions, exerted by bodies pulsating or oscil-
lating in a fluid medium. We owe our knowledge of this branch
of Eydradyaamics chiefly to the two Byerknes, father and son.*
They have shown that bodies pulsating or oscillating synchro-
nously in.a liquid attract or repel one another cenendinae on whether
they pulsate or oscillate in the same or opposite phase. Further-
more, these bodies set up lines of flow in the liquid, real hydro-
dynamic lines of force, which simulate exactly the lines of force
in magnetic or electric fields. The sign of this force is, however,
in general, just the reverse of that in electric or magnetic fields.
Bodies pulsating synchronously and in opposite phase repel each
other, although the form of the jie old the y produce 1s 1dentical with
amerocenmnlibe magnetic poles which attract each other. Simi-
larly, two spheres oscillating synchronously and in the same phase
repel each other, elu they too produce a field like that
between opposite magnetic poles. The following experimentally
derived figures, taken from Byerknes’ text-book, illustrate this
identity of form and reversal of sign in the electric and hydrodyna-
mic energies:

6 See Hydrodynamische Fernkrafte, v. Bjerknes; 2 vols. Leipzig, J. A. Barth, 1902.

30 Arthur B. Lamb

a AR) XX
PATA AWA
| 4 ee

Fig. 1 Lines of force between unlike magnetic poles. REPuLston.

\ i ! pe - ° : t !
\ Sel f ae nee ‘ ; ha /
= / y
= \ . \ ee Sy \ \ ee yi a
Ny
22 Ss a a ea a
Se ==, ba aa a
= ee! = See —_
SO St ee ad
7 Zi
Dae ae Ne ee es N
eee sf NR <7) iK , S
Games {'\ NX Se, \
7 ah .\. Se fon
tite ‘ 0 : ‘\
\ Lae / \ \
/ y \ ‘ i ee C t \ \
! ean . Z, f '

Fig. 2 Lines of flow between oppositely pulsating bodies. Atrraction.

It is this exact reversal of sign of hydrodynamic action at a dis-
tance, as compared with electric and magnetic actions, which
makes this force peculiarly applicable to the case of mitotic figures,
obviating many objections which beset the previous explanations,
and particularly the fact that with previously considered polar
forces if one made an assumption which would explain the form
of the field, the motion of the centrosomes appeared contra dictory

The Mechanics of Mitosis 31

and vice versa. If, however, we assume that the centrosomes are
pulsating synchronously and in opposite phases, or oscillating
synchronously and in the same phase, we obtain the desired repul-
sion, and at the same time we get mitotic figures corresponding to the
configuration of the lines of magnetic force between opposite poles.
That is, we get a configuration of spindle fibers and astral rays
precisely like the actual ones.

The cases of tri- and multi-polar spindles, so difficult to explain
on electrostatic grounds present much less difficulty here. If each
centrosome were oscillating along a path radial to the common
nuclear center and in the same phase, mutual repulsion, combined
with the proper configuration of the astral rays, would be obtained.

The movements and configuration of the chromosomes are also
better explained on hydrodynamic grounds than by previous
assumptions. It is not even necessary to assume that they execute
any independent oscillatory or pulsating motions. Byerknes has
shown that bodies suspended within the field of force of oscillating
or pulsating bodies are attracted or repelled depending on whether
they are lighter or heavier than the surrounding medium. ‘This
attraction or repulsion is due to oscillations induced in the sus-
pended bodies by the permanently oscillating or pulsating bodies.
The chromosomes, if heavier than water, or the cell fluid in which
they are suspended, would be repelled from each centrosome and
would come to occupy a position midway between them in the
equatorial plate. Moreover, they would not move outward to the
boundary of the equatorial plate. ‘Their induced oscillations,
though repelling them from each centrosome, would attract them
toward each other, and this action would tend to keep them in
the observed axial position. If the chromosomes should become
lighter than the cell liquid, the repulsion from the centrosomes
would change to an attraction. ‘This immediately suggests that
it may be simply a change in specific gravity of the chromosomes
which causes them to diverge, after splitting, back toward the cen-
trosomes.

[t is now of interest to inquire whether this hydrodynamic action
at a distance could possibly be strong enough to account for the
actual movements of the centrosomes. It is, of course, almost

32 Arthur B. Lamb

impossible to decide this by calculation, in the present state of our
knowledge, but if we assume that the centrosomes are smooth,
hard spheres and that the cell fluid is homogeneous and as mobile
as water, it is not difficult to calculate how vigorously they must
oscillate or pulsate in order that they shall move apart with the
observed velocity.’ Taking the radius as 0.0002 cm., the dis-
tance apart as 0.003 cm., the time required for this maximum
separation of the centrosomes as fifteen minutes; if the amplitude
of oscillation equaled two diameters, the frequency required would
be 2000 oscillations per second; if the amplitude were eight diame-
ters, the required frequency would be 100 oscillations per second.
With similar dimensions, if the centrosome pulsated so that its
greatest volume were three times its least volume, a frequency of
some 130 pulsations per second would be required.®

These frequencies are greater than one would expect. They
do not, however, involve any great linear velocities, for the dimen-
sions of the particles are very small. ‘Thus the frequency of 2000
oscillations to the second only means a linear velocity of the centro-

7 The formula of Stokes
F=6zpur
(Brit. Assoc. Report, p. 445, 1887) applying to the motion of spheres though viscous media was used
to determine the force needed to give the centrosomes the observed velocity. To find the needful fre-

quency of oscillation this was equated to the expression

6 a
: 47d!

derived by Bjerknes for the attraction or repulsion between oscillating spheres.

Similarly, to find the needful frequency of pulsation it was equated to the expression

a a)

8/9 — (a°— 1)? p?

also based on a formula derived by Bjerknes. In all of these expressions r represents the radius, d the
distance between the centrosomes, /t the coefficient of viscosity of the medium (water), p the frequency,
S the “action moment,” v the velocity, and a the ratio between the mean and the maximum radius of
the pulsating sphere.

8It might also be pointed out here that similar calculations on the hypothesis of electrostatic action
show that, if the capacity of the centrosomes is simply that of conducting spheres in an isolating medium,
a potential difference of nearly two volts would be required; if the capacity is that of spheres surrounded
by a ‘Helmholtz double layer,” a potential difference of only a few thousandths of a volt would be

necessary.

The Mechanics of Mitosts 33

some of 2 cm. per second. It is, of course, almost impossible to
say what effect a viscous, heterogeneous field would have upon the
calculation, so we are obliged to leave the quantitative side of the
question as quite unsettled.

Besides the oscillatory currents produced by pulsating and oscil-
lating bodies, Bjerknes has shown the existence of steady currents
in the fluid medium toward and away from the centers of motion.
Similar currents have been observed in dividing cells, particularly
centripetal currents between the astral centers. It does not, how-
ever, seem wise to treat this or similarly less pronounced phe-
nomena in our present state of ignorance.

The assumption of a pulsating centrosome or centrosphere is by
no means an impossible one. The assumption of an oscillating
centrosome is not even improbable. The assumption of syn-
chronous pulsations or oscillations involves no mysterious synchro-
nizing mechanism. Random oscillations or pulsations would cer-
tainly tend to become synchronous by mutual interaction, while
after the closed spindle fibers had formed, whatever their nature
may be, any other rate of oscillation would be very improbable.

The fact that such oscillation or pulsation have not been de-
scribed is not conclusive.

Our knowledge of the subject is based almost wholly on dead
material, and moreover the oscillations and pulsations may be
very rapid and small.

In conclusion, I would again like to emphasize that the above is
nothing but an ad hoc constructed hypothesis and intrinsically
therefore only of hypothetical value. If, however, it calls atten-
tion to a little known phenomenon or stimulates fresh observation
it will have served its purpose.

CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORY OF THE MUSEUM
OF COMPARATIVE ZOOLOGY AT HARVARD COLLEGE. E. L. MARK,
Director. No. 193

THE REACTIONS OF PLANARIANS ITO LIGHT

HERBERT EUGENE WALTER:

Witu Fourteen Ficures

Mp te Od CEO ML tejasetlcrassy sieve e/ave os stase aves ste aie soe a1els ate etre areloye avatecera leis evainiereinnsraicaete o eeioreele 37

UG MELISCOLIGAL faetercyatsfetctereroieialeislele eictal-isyaio nreiercte HopduacpaduanducooapoubooD Adasen ousbabop 38

SVT Gee Milter l mere etetey terisiozestortaresetovecoxeiais )caayoictetere cle st cteiete sisiersss/ayesctersrereicieieiarelnere erates sie eteteieee 45

Va Criteriaitormedsuring bel avion ves deratsei= stelaeistars steisiclorsietel cereale rater ae 47

WA © Dsery cl tloriswreretoyesetes staves tetetet rater cie/crsie ese vereiste lvls) s]aisve: «vial crstatactote rents orate cleaver nee cheer 49

Hime E Told) stl Sycooodo o GUD RUE Aa Dao no Reon aUraeacoaed se dopendoceodernadaacassad 49

. LX. AUDEN iO dteh Cela Sapo hbenaeroTgnbenElsiua GoctnceonucdeonocbTd 66500000 50
Rate OfllocOmOtlOnns fatarsiniesctctors oscle) oferctarereyereasieraect=tale ae tereie rarest 50

. ARG bhy355 dap oe on DpH eR pAarAUAehEEnEddutechododacucnootoapeds SI
| (ChEpTTSO Ichi sone opodbentandonpaavadpeooduMdcdaasavconDes 51
SUMMA reine lars sisinisia\cisicls oy=taiere oie'aYelaye\ ic /aasysiejaicretratvstcictetetetbersieteicle sr

Biss Non=directive light -jsr<\ays/ats ters e)ars(ti 2istsyaistsracei nel te/eicterniclerels\emmiale sereieeie elec 52

Gam AD Paratusara str cecisseleitectciersteleiswiois a cleletsemtanteinet perenne ears 52

De Results Serer rstate;oss,aforesajotelasyalotclaters ayars 2ts1saje eictaieysleisiotsie ste/stanietersteteie aceite 56

IRafelohlocomotioDy «ssh tsisiate/siets vieltersssierars Yeeieceineier eos oeiee eee 56

Alin sth eae ho ceonnotno BouaeRqoEnDooEsmErndanusscoodadsodncuac 58

GChanpeahcourse. poate steletererelals.erais Actors atersthslefeetane eter ere ee eee 59

Beeps ACE srornistassisielaie elctersTasa(eletedate:nialetai Taya os aise oes tiers nrateleraetaectioilerste 64

SUM Maryietatersiancelaterictaists ofeletateetaitearcta cistercierercie etsvete lettereietacsteereterate 71

PRRENOLOCAKIS ARB defector ttcracier ryt cteteve or SS Se Sat ate a ee Tae in eee 72

Avemuconstantidirective lighteacs tists da stetaetditerisicliis ecto seletem ceiseten tetas 72

Orientation seer iceciocctietorsmtere oti eislercareieiereis revels reenter eeicrornieracntiets 72

IRACG OF LOCOMOLION sjeysielcscjerofsiscorsisielore sfercisinicietelcistann eieieteistersinletele ete orale 75

Ghanpeanicharacteroficourse seins ivi-rsicieis sin arsercisierarsiorsieteleeicierersiereve 78

Prcetiracviohorienta tons sre iejeleister-infeci= ce acieie cisistalcieisiciciarsie’s sleistelerevareie 719

IDR ORANG Hioht-adpotde send sodanbarenbasdcnontusaodoedeouua 80
Durationiobiactivityatectuisnviesiccisene einicnis cietsven elatcieciace ice sm ace 81

= Mimeixequired tolleave a mmitcircle. --.icisj-islcidiels cc slalsieis cis ervicecsntys 82
Manner of coming to rest 83

SSN Ea Goh St gonbeko Apopoud oh doen aoanAneUe COUN oneNo ene sea ne 86

Journar or Exrerimenrar ZooLocy, vor. v, No. I.

Herbert Eugene Walter

B; In.changing: directive laghes <i... 1<t-)-)-1=/o)z)atels/=1=1=/=1al= stele = sl =\oic/etelelotola/eeraiete teeters 87
(Ghanges imlintensitye:jetssce! a= o\aye.c rer tereicssis iotetels sinislessfelonateteteis1<eteyeistere 87
GhanpesimdirectiOninse aoe remiers ce selec s eisia eee n/heioie eters 88
Ritinlol iy sap enna AED Aan DG IuobaupaenandoraaragdaosouAnoDaccda 89

C inycombinationwwith'other responses: <1... .e.0.)nis ins sleleieisinicisie'e.aislsisizisietaisfeieels 89
(Esai Fs shedhoasdaor coded as muMaAadbosbn bocobcunonoDonNAsDOSNUGd go
PHI SMOtaxis eecicciai eer eras crohenateletaietapetcte ctataleiste atersiatel= atateraes aceeterete gz
Goniotaxissrn..-. «licence Nels e slsesicisier/aee setae see eee eee eile 94
Chemotaxis es vous cctasickes tele eior acetals semaine stratermtelet aces 94
SUMMA YG rors arctasstolecs Grek opatalarevainioleiers tistencte ave otesstoloatet atetete @teteyose 96

gy lRinds/of behavior: joer ace ceiseniee is ae aioe aie. cioisioe sisiseidke See ei stole otete ers sepieeis 97

A (Genencjand:specifie behavior jateressvcreoicretet sry -\alossloles ola lose = ei ele/alevetetaye ateradsfeleiat 98
Percenta re obmegatrveresssistetsiete sta (oy: che utalotelate shetelsieveletateletates tetsteneiets 98
Character of the course in directive light..............0+eeeee0ee 99
Duration of movements (ris sis:aicictetayere l= afa'= ie (ele c's/n)sis/ayeraters =tetslols/oreiare 105
De greeiOl wandering states cic ssotaias<tesetersieisiete aletsiele! sto)<\s ojeleterepateieretetiata 106
Rate of locomonons 4 2.7 << cersierss vives once ¢ vis he vivieue sre mieisieue ieee 107
Tuimexequired;tolleave:aimniticurcles <ste:aiaresets areal= siacecereieleyaclae elem 107
The effects of fatigue....... Rasen TadpeorniadacmianaaicGecnttads 108
Responses to chanpesmintensitycncclaan siete eels sieleholeite ieeereieetel ere 109
Manner ofl coming tonrestisysisjsc/stsrepac se cise ain ctere oratahetetefenstevaystelsteerate 110
Bint ty cor Sen on non EBL on qOscao CeO EEOcabHoInohbeacundoc 110

IB Individwallbehaviorgencyetascissleterie selon <saieeicis sls oslo siet-toisiers reer enter III
‘Rate omisuccessive sd ayss..c dois + sic151s susie, ef- os) arorerenaieto sovatersraiatetetstotniate 112
Relative value of individual behavior.............-+0+seeeeeeeeees 113
Avcave: planariam' <7. cli-tsyatt a/atels niotels c/a stole ete nese ieee eet oars 114
Rilke s Sanco A HOnHOOUS HAC OnronneEnOdD oo Uanomasn dodoncoonss 116

(Pages 117 to 162 are printed in vel. v, no. 2)

4. Basisiofibehaviorss2cescicons-seete ees ane aves aint ee Cine CeaieT ee eee rete 117
A” Morphological’ basisiofibelnawior.-/crcc,ct01</=:e:cjes!/apa)s'a(e =! ntela(o/o\oaielstelsteievala}eetesrataye 117
a), General! formof the bOdy ss sic. s:esseis:thate-<:t1n's1a/e'el<ie'ace siete eareistersteratsitatelenciele 118

be (Photoreceptorss sewiqseectcsecice/icle ois elienryteleieteisya staretstetterctee cre rene 122
SUMUMAY ses aterctercloyaje ceislelaeie ss elie neste eeimetteietelererere sentir 127

B Physiolopicalibasis of behaviors tater. slois/ateieiale vl eyetstetereteyoieteletais vet stersfofetatstere ateiete 128
a Classification of: physiological’states/.c%.. wei vieleloaus aiele ese rlerersieie rete ster 129

b Changes in physiological states induced by light..............+.+s-0005 130

Effect of different intensities i.e: ejere:ctetois: store laietn tots oretetstestet teat etateteeen 130

Effect of'excessivellight: mimesis el slekeielalseiieveloeierileeieie eitater (eters 131

Effect of sudden change in light conditions.............++s+eeeeeee 131

Effect of continued exposure to light............-6 0.0 eee ee seen eee 131

Effect of previous ex posure:to)d ark: jelsteiers\stele ator ster te siatatsteteteperetsr ictal 132

ELCs iqgnappnnodacuacncnuoudan dscancu Joona osaecesteoee 133

G@: Psycholopicalibasisiof behaviors. eve peyre/eseletetatstejesstar ste interac eter etniens oe felotereratofonste 134
a, How,muchican'planarians)see' tise eicje siete! =rasetat=\ sietele)s(aiein e\efeleterers s7els\alele|</s 134

b: “Areiplanarsans/ablejtojlearm ? rarelateetersjereteters tet leterslete ele iets/aratelet=te\e\ele late? 135

The Reactions of Planarians to Light 27

Vili General: Conclusions fare <ls/slats(olayar/afols\elarer/=\elsta\elelatolaIs\ele\e7e dbopondonuecvonooacoRaass ae 138
He PD IFeckOn OLMteL sity jo/ate ys <lnysieteictoi<isl te = 1m eleialarelase]sictelse EES as cbatete ohessi seaponstetsvaae es creieya se 138

Age Historicalusetcmstacpicaste stich ererietec cient viachavsicts eferecitie eerie aie cetarssretes 138

B; Conclusions;with reference to;planariansicc. cic cee sc sence meine weog asincice vee 140

a The distinction between direction and intensity..............--..+2455 140

b “Lhe modifying, influenceiofidirettOn sis. ccicis:= «0 ce wie ee ee rieie cide eee 141

c Instances of behavior due to intensity alone ...............+--+---+00 142

d Theimoditying effect of other factors<.. 5. (-..-..6ce:cree nial @ sieinieisisie anise 144

Wuininih 97 sone vaccndades Some mag tnaUNoh 4 one ssocsDomsesenoon 145

PB aictllriveliGegim@relaa}y hint aohconoshoe codon apNBesResaaamasnopacnpocaDesboodpo 146
Siininkla Honshoshossouppocptuponvedeadtuparnouscacscoconocotc 153

€) YE eit caoo fo cosciponnoddadobenongon dob ocpnunaunocpbos house deeHoGnuCEos 153
Suing gusnp esac ana oo too sbunpEbenDo cH sonmpood CoD doc dao! 155

Wat Tyo) Hoya doh Mabe onaenon bao odparodoesssvenootodnonud BODO SNGOBRAD EAD nOSROCaoUs 155

I InrrRopucTION

Light is one of the physical factors which influence the behavior
of organisms. ‘The great majority of living things are normally
subjected to regular periodic changes in the amount of light to
which they are exposed during the alternation of day and night.
In addition to these constant periodic changes, there are innu-
merable irregular gradations in both the intensity and the character
of the light naturally acting upon any organism. An agent of
such wide range and almost universal influence as light ought,
therefore, when properly analyzed, to prove of material service in
interpreting the behavior of animals and plants. The depend-
ence upon light of animals provided with organs of sight, is self
evident. ‘The direct bearing, too, of light upon chlorophyllaceous
plants in the manufacture of their food substance, is plain. But
how far light plays a direct part in the life of non-photosynthetic
plants and of animals which cannot “see,”’ is less clear.

Although possessing eyes, it is very probable that planarians
are unable to see in the sense of distinguishing shapes, and it is
questionable how far they can distinguish between even large
regions of different light intensity.

The object of the following paper is to examine the relation of
light to animal behavior as applied to certain planarians.

38 Herbert Eugene Walter

Il Hisroricar

Our knowledge of planarians, as of most other animals, has
passed through certain historical phases, during which emphasis
has been laid first upon taxonomy and anatomy and latterly upon
embryology and zoogeography. The results of these various
forms of investigation are highly important since they make the
foundation for all future work upon this group of animals. They
have, however, only an indirect interest in the present connection
and do not, therefore, require review.

Perhaps the most modern advance in our knowledge of pla-
narians is represented by the school which treats of them as living
objects whose individual behavior is to be intimately correlated
both with their structure and environment. ‘The most noteworthy
contribution from this standpoint has been made by Pearl (03),
who has analyzed in considerable detail the reactions of fresh-
water planarians (notably Planaria maculata, Planaria doroto-
cephala and Dendroccelum lacteum) to various stimuli. He has
not, however, discussed the effects of light except incidentally.

The earliest reference to the relation of planarians to light is
by Dalyell (’14). In his interesting volume on planarians a great
number of keen observations upon the general habits and struc-
ture of planarians are made, which have since been confirmed,
together with certain statements which have not fared as well with
the advance of scientific knowledge.

He makes the statement (’14, p- 9) that “most planariz court
the light indeed;' but P. flexilis rather inclines to shun it, less, we
may conjecture, from being warned of its presence by the specks
or eyes, than from some disagreeable sensation produced on the
body.” Again, referring to P. felina (’14, p. 46), ‘“ This planaria,
like the rest of its genus, is powerfully excited to motion by the
presence of light. If a number be confined in a glass vessel, the
whole assemble in a quiescent state, on the side next the light It
is a little surprising that Dalyell should have received the impres-
sion that the majority of planarians “court the light,” since he
clearly points out the nocturnal habits of these worms. He

1 The italics are mine.

The Reactions of Planarians to Light 39

doubts whether the eyes are of service in finding food and says
of worms under aquarium conditions (14, p. 107), “If remaining
aconsiderable time unchanged, the planariz decrease more rapidly,
they become languid, scarcely moving either by the influence of the
light or heat, and at last adhere entirely to the side of the contain-
ing vessel, where they perish.”

Dugés (’28) observed that when light 1s concentrated by means
of a lens upon either Dendroccelum (?) or Planaria, movement
results which is most pronounced when directed toward the ante-
rior end of the worm. He tested the effects of direct sunlight and
of diffuse daylight as well as of candle-light, and concluded that
the response increases with the intensity of the light. The non-
dioptric character of the eyes he has described remarkably well
for one working so long before the days of the microtome, and his
conclusion, already suggested by Dalyell and later confirmed by
Kennel (88), and others, that the eyes play no part in the finding
of food, is noteworthy. He also notes that planarians seek the
dark.

Dalyell (’53, p. 99), in a later volume says, “On April 29 I pro-
cured a fine specimen of Planaria cornuta, which spawned soon
afterward. The spawn had been breaking up for two or three
days preceding May 24, when multitudes of extremely minute
yellow specks were seen swimming in the water. Their motion
was sufficiently active, without being very quick; it was pursued
in all directions and the spawn being contained in a small cylin-
drical jar, the specks crowded to the sides next the light whereon
numbers remained almost stationary.’’ Again (’53, p. 104),
“When withdrawn from the dark the young Planariz rose in great
numbers toward the surface of the water, congregating on the
sides next the light.”” It is extremely doubtful whether the organ-
isms here described were really young planarians. It is more
likely that they were the young of some other aquatic animal.
Dalyell correctly describes Planaria lactea (Dendroccelum lac-
teum?) as being nocturnal. He observed that numbers of this
species, beginning activity in the evening, rose on the sides of the
jar, although many had descended again by morning.

More recently attention has been specifically directed to the

40 Herbert Eugene Walter

light relations of planarians in various papers by Loeb, whose
important contribution in 18go, ‘Der Heliotropismus der Thiere
und seine Uebereinstimmung mit dem Heliotropismus der Pflan-
zen,” paved the way in general for all work on this subject. He
found (’93b, p. tor) that Planaria torva is not “‘heliotropic”’ in
the strict sense, but rather “unterschiedsempfindlich,”’ that is to
say, it did not always move away from thesource of the light in the
direction of the rays and remain as far removed as possible, but
moved about more or less at random, coming torest in some area
of lessened light intensity.

In a later paper Loeb (’94, p. 255) states that when planarians
are suddenly brought into the light they begin to move, an increase
in light intensity leading to activity and conversely a decrease, to
rest. [he grounds for this conclusion are not made clear. He
further confirms the view that planarians are active at night, com-
ing to rest in situations of lowered light intensity in daytime.

In further experiments P. torva when decapitated was found
to react to light precisely as normal worms do with the difference
that the reaction required more time. Thysanozo6n brochii, a
polyclad, on the other hand, lost its power to respond to light when

“the eyes and brain were amputated, from which Loeb draws the
conclusion that animals which are closely related morphologically
may exhibit wide physiological differences.

Hesse (’97), in his classic study of the anatomy of the turbel-
larian eye, mentions some experiments and observations on the
behavior of planarians and in addition makes valuable contribu-
tions to the morphological basis of light reactions. He observed
that planarians become active at twilight and he also experimented
upon decapitated worms, apparently without being aware of the
previous work by Loeb, with whose results his own agreed. He
found, as Loeb did, that worms deprived of eyes finally come to
rest in areas of lesser intensity much as do normal worms but after
a longer time. Hesse found, too, that Dendroccelum lacteum
came to rest in the dark 119 times out of a possible 120, whereas
with Euplanaria (Planaria) gonocephala the same result was
effected in only 88 out of 120 times, notwithstanding the fact that
the latter has more highly developed eyes than Dendroccelum.

The Reactions of Planartans to Light AI

This led him to state (’97, p. 552) that “die Starke der Reaction
auf Lichtwirkung nicht der Starke der Lichtwahrnehmung ent-
spricht,”” and he ascribes this difference in behavior to a differ-
ence in the ‘Gefihlston”’ of the two species. Amongother observa-
tions described by Hesse, the two following are of importance
in this connection, namely, that a sudden introduction of light
caused an almost immediate turning away on the part of the worm,
and, that worms with eyes could not be made to remain in the
light when escape was possible. In his opinion this apparent
perception on the part of the worm is due not to the animal’s
ability to distinguish light but rather to unpleasant chemical reac-
tions set up within the organism as the result of light stimulation.
And, lastly, Hesse showed that the general position of the eyes
of a planarian, together with the arrangement of their sensory
portion, partly enclosed as it is within pigment cups, affords a
device whereby the worm can be oriented to light. By means of
this simple apparatus it receives a localized stimulus, which enables
it to distinguish the direction from which the light comes. If
light, striking the eye of a worm, fell-upon sense cells which were
unscreened in any way by pigment, there would result a general
stimulation without localization of the stimulus and consequently
orientation could not be effected.

Parker and Burnett (00) sought, by quantitative methods and
with more accuracy than Loeb or Hesse, to establish the part
played by the eyes in light responses of planarians. They came
to the same general conclusion as these authors since they found
that Planaria gonocephala without eyes reacts to light essentially
as normal animals do, except that the reaction time is somewhat
longer. They also showed that worms when pointed toward the
source of light travel at a slower rate than when headed in the
opposite direction. With regard to the mechanism of the light ,
response they say (’00, p. 383): ‘“‘We have seen nothing in our
experiments that supports the opinion suggested by Hesse (p. 551)
that reactions such as we have described are due to the direct
influence of light on the internal parts of the planarians, and
we are more inclined to the view that these reactions are initiated
by the effect of light on the integument of the animal, 7. e., are

42 Herbert Eugene Walter

due to what Graber (’83, p. 229) has called a ‘dermatropic’
function.”

Bardeen (’ora, p. 13), speaking of Planaria maculata, states that
“susceptibility to light is apt to become lost if worms are kept in
captivity,’ and he notes the fact, already brought out by Chichkoff
(92), that pigment becomes reduced in sunlight. Hesse had
previously emphasized the point that the pigment of the eye of any
organism has in itself primarily nothing whatever to do with light
perception. Bardeen further found that small pieces of planarians
capable of locomotion will respond to light in the same way as
uninjured animals, and he notes (’o1a, p. 13), that the worms
seem “to move about more by night than by day.” In a later
paper (’otb) he speaks of the fact that when a dish containing
planarians is brought into light the worms are commonly roused
to activity, although how far such activity is due to light and how
far to mechanical disturbances he does not make clear.

Lillie (’o1), experimenting upon the regeneration of Dendro-
coelum lacteum, discovered that posterior headless parts fail to
give the typical reaction to light and are incapable of regeneration.
He draws the conclusion (’or, p. 132) that “any symmetrical piece
of Dendroccelum capable of regeneration tends to come to rest in
the shaded part of the dish precisely like a normal individual” and
that parts incapable of regenerating ‘‘also become incapable,
after a day or two, of performing the usual reactions to light.”
These results on Dendroccelum, it will be seen, are similar to those
Loeb obtained in experiments upon the polyclad Thysanozoon.

Curtis (’02) reports from laboratory observations 42 cases of
fission in Planaria maculata, of which number 39 occurred between
Io p.m. and6a.m. He adds, however (’02, p. 524), that “this
did not seem due to the amount of light to which the animals
were subjected during the day, for some of the dishes were so
shaded that there was practically no light, day or night, except
when they were being examined, and the division was the same
in these as in others which were exposed to full daylight.”” A case
of division in Bipalium also occurring by night is described by
Lehnert (91).

In a contribution to the geographical distribution of Planaria

The Reactions of Planarians to Light 43

gonocephala, P. cornuta and P. alpina,*? Voigt (’o4) incidentally
refers to the manner in which these animals come to rest in the
darkest part of a dish. He afhirms that when an aquarium is sud-
denly lighted at night only those that are hungry, :. ¢., those with
comparatively empty digestive tracts, are found in motion, and
he notes that in certain conditions worms may remain quiescent
for weeks. The statement made earlier by Duges, that the eyes
of planarians play no part in finding food, Voigt confirms. ‘These
organs he explains are an aid in distinguishing differences in light
intensity as well as the direction from which light comes but are
entirely incapable, owing to the simplicity of their structure, of
discerning the form of objects. In his opinion worms crawl into
hollow stems and similar sheltered places to escape light rather
than for warmth, as Wilhelm: ('04) suggests. Neither author,
apparently, considers the possible part played by thigmotaxis
under such circumstances. Of the delicacy with which worms
react to light Voigt says (’04, p. 173): “Die Empfindlichkeit der
Planariden gegen plotzliche Belichtung tritt so scharf hervor,
das sie fiir den Unterricht eines der anschaulichsten Beispiele zur
Demonstration der Lichtflucht bei niederen Tiere darbieten.”
Notwithstanding this high degree of sensitiveness to light, he finds
that the worms when seeking their food leave the shade and come
out even into direct sunlight. And, finally, concerning the bearing
which light has on the problem of distribution, he concludes (’04,
p- 175): “Auf die Verbreitung im Allgemeinen hat die Belichtung
der Bache wenig Einfluss, da sich in der Regel genug dunkele
Schlupfwinkel finden, in denen sich die Tiere verbergen konnen.”’

Darwin (’44, p. 242) observed that /and planarians, “especially
Planaria tasmania, had an immediate apprehension and _ dislike
of light, which they showed by crawling, when the lid of the box
was taken off, to the under side of pieces of rotten wood,” and in
his enumeration of the places where various species of land pla-
narians were found, their avoidance of light is plainly shown.

A note by Leidy (’58) refers to finding Rhynchodemus sylvati-
cus crawling about on fences frequently at night, but rarely by day.

Pp. alpina = P. torva according to Borelli (’93).

44 Herbert Eugene Walter

Moseley (’74, p. 111) states that “land planarians are probably
all of them nocturnal in habit.” Speaking of the Ceylon land
planarians in particular he says: “They are found in dark places,
such as under large fallen leaves, and in confnement they coil
themselves up away from light.’’ He mentions also the fact that
Planaria torva and Dendroccelum lacteum choose the dark side
of the vessel in which they are contained.

As has already been mentioned, Lehnert (’91) found Bipalium
kewense undergoing fission in the dark. Both Bipalium and
Geodesmus, hee says, seek continually to hide in shadowy places
avoiding even diffuse daylight. Concerning the degree of light
perception possessed by planarians, he offers the opinion (’9I, p.
326) that ““Bipalium scheint mit seinem Augen die Umrisse von
Gegenstanden in Lichte wahrnehmen zu kénnen.”

Hogg (’97) notes that Bipalium is nocturnal in habit, remaining
pieae during the day.

Only Fiseiel references to the polyclads are found bearing
upon the question of light reactions, as for example this sentence,
which occurs in Lang’s exhaustive monograph (’84, p. 641), “Die
meisten Arten scheuen das directe Sonnenlicht.”’ “The behavior
of Thysanozoon with reference to light has already been mentioned

(Loeb, ’94).

Concerning the light reactions of the rhabdoceeles, especially cer-
tain green fone 4 in Shih the green cells are probably symbiotuc,
a considerable literature may be found. ‘The principal papers
relating to these forms are as follows: On Convoluta schultzii,
by Geddes. (79), Barthélémy (84) and Delage (86); on Convo-
luta roscoffensis, by Haberlandt | COL), Bohn: Coza, (o3b, Ose);
Gamble and Keeble (’03) and Fiihner (’06). Vortex viridis and
Mesostomum viridatum (?) are discussed by Schultze (’51),
von Graff (’84) and Sekera (’03). A résumé of these papers is,
however, out of place here, since the presence of green cells in the
organisms involves an entirely different problem from that
which is under consideration.

The foregoing historical sketch furnishes the basis of the follow-
ing general summary of facts which have thus been established with
more or less certainty regarding the reactions of planarians to light.

The Reactions of Planarians to Light 45

1 Planarians are nocturnal, seeking the dark when exposed to
light.
2 The eyes are useless in finding food.
3. The anterior end of the body is the part most responsive
to light
4 Decapitated worms act normally except for a slower reac-
tion time.
5 Onientation to light depends largely upon the character of
the pigment cups of the eyes.
6 The relative energy of the response is dependent upon the
intensity of the light.
7 Pigment is reduced in sunlight.
8 Pieces of worms which are large enough to move or regener-
ate react to light.
g Fission may occur more readily in the dark.
10 Different species respond differently to light.
11 Light reactions diminish during “captivity.”
12 Planariansare “unterschiedsempfindlich” instead of “helio-
tropic.”

III Marertar

The species principally used in the following investigations
were Planaria maculata Leidy; Planaria gonocephala Dugés;
Phagocata gracilis Leidy; Dendroccelum lacteum Oersted; and
Bdelloura candida Guard, all of which are inhabitants of fresh
water except Bdelloura, a salt-water species, found living semi-
parasitically on the horseshoe crab (Limulus polyphemus). Some
observations also were made upon a cave planarian, that as yet has
not been identified but which may belong to the genus Phagocata.
This interesting worm was kindly placed at my spose oy Dr:
A. M. Banta.

At any season of the year an ample supply of fresh material was
easily obtained except in midwinter, when it was necessary to cut
through the ice and dredge up from the bottom water-weeds to
which the worms cling.

The source of supply for Planaria gonocephala was a small.
pond to the west of Fresh Pond in Cambridge, Mass., while Pla-

46 Herbert Eugene Walter

naria maculata, Dendroccelum and Phagocata were chiefly obtained
from a pond at Falmouth, Mass., where they are especially abun-
dant. ‘Twice, through the kindness of Professor Parker, aquaria
were generously stocked with Dendroccelum, from a spring on
Mount Monadnock, N.H. Bdellourawas obtained from Wood’s
Hole, Mass., during the summer from freshly caught horseshoe
crabs and, later in the year, from specimens kept in captivity.

The setting-up of balanced aquaria in which planarians would
thrive did not prove to be a difficult matter. The following
method, based largely upon suggestions by Wilhelmi (’04), was
used. Jars were filled to the depth of two or three inches with
cinders, dirt and dead leaves, over which was spread an equally
deep layer of clean sand. Clear water was then poured into the
remaining space and the whole allowed to settle, after which afew
such plants as Anacharis or Myriophyllum, with whatever micro-
scopic life might adhere to them, were added, together with a hand-
ful of large pebbles to diversify the bottom. ‘The jars were kept
covered from dust in a cool place and occasionally a crushed snail
was dropped into each one to supply the worms with food.

Planarians require pure water. Whenever for any reason the
water in which they are kept becomes foul they will desert their
places of concealment and crawl up the sides of the jar, while
water that has been standing in lead or iron pipes quickly causes
them to disintegrate. Rainwater or water taken directly from
some natural source, gives better results than that which has been
conveyed through pipes. Naturally the least chemical disturbance
takes place when the worms are kept in water dipped up at the
time and place of their capture.

Planarians will live without being fed for over three months
when isolated in jars containing nothing except pure water, but
meanwhile they decrease regularly in size. It seems to be impos-
sible to “starve” them in the sense in which higher animals may
be forced to die from lack of food leaving behind a dead body.
These worms instead simply consume their own substance almost
to the vanishing point.

During a part of the summer of 1905 observations and experi-
ments were carried on at the laboratory of the U. S. Fisheries

The Reactions of Planarians to Light 47

Bureau at Wood’s Hole, Mass., and I wish here to express my
thanks to the director, Dr. F. B. Sumner, as well as to others in
authority there, for their uniform courtesies. The bulk of the
investigation, however, was made at Harvard University. I am
deeply indebted to Professor Mark for the privilege of having
a place in his laboratory and particularly to Prof. G. H. Parker,
under whose immediate direction the work was done and whose
daily counsels and generous suggestions were indispensable.

IV Crirerta ror Measurinc BEHAVIOR

Both the form and the structure of an animal set a limit to the
character and degree of its movements, which no combination of
stimuli, external or internal, can force it to overstep. In estimat-
ing the influence of light upon planarians, therefore, it is necessary
to know not only the normal behavior of the worms but also the
possible range of their reactions under any circumstances. For
example, the ordinary gliding locomotion of planarians is accom-
plished by means of cilia beating in a mucus track and augmented
by muscular contraction. It is physically impossible for this
sort of locomotion, even under the most favorable conditions, to
exceed a certain rate. By the use of excessive stimuli, however, a
worm may be forced to abandon this accustomed gliding for a
somewhat faster method of progression known as “crawling”’ or
“humping,” in which the muscles are used more than the cilia.
But when this is done the limit of possible rate of locomotion has
been reached, at least for fresh water planarians, which cannot
be urged to abandon entirely contact with some support and to
swim freely in water, although the marine form, Bdelloura, does
have this addition to its repertory of behavior.

The following observations may illustrate more specifically what
is meant by range of behavior. Planaria maculata, when gliding
on the bottom of a dish, was lightly touched on the anterior end
with a hair mounted on a glass rod. During one hundred trials
of this kind eight different responses resulted, which may be indi-
cated as follows:

48 Herbert Eugene Walter

Times

1) | Contracted wand turned) asi Garerctetrs:sccxsieteyeraressictesest1oia/ sissies ets ofeiars stat siayarets aistelatet atest steyetateteneterete 32
2 Contracted, lifted up the anterior end, and turned aside........-....0+eeeceeeeeeceeccece 27
3 Contracted, lifted up the anterior end and went straight forward ...........-.......+se005 17
4 Contracted momentarily and then went straight ahead .............020.020seeceeeeeees 5
5» Didmot,contractibutiturmed asides a. cccreerae sclas ace -acigad meine eee CPE ere tEee 2
6 Did not contract but lifted up the anterior end and turned aside..........-.0.00eseeeeeeee 7
7 Did not contract but lifted up the anterior end and went straight forward.................+ 9
8 Did not contract but went straight ahead...... IPR ACO NOD Onan dopeTctn cacdacoacdad I

DO tal sicrerayats store state senceloepeteite re tetogen Me oteis lel stoi sveie/o\s0ore co, 312 agsibia, nia sia) el ofer ticle Meiers tear eee meteteore 100

Animals which, like planarians, present a limited range of be-
havior are, therefore, more favorable subjects for experimentation
than higher forms whose structural complexity increases their possi-
ble responses, making in consequence the analysis of cause and
effect in their activities more difficult. It is evidently desirable,
then, to have as many different ways for measuring behavior as
possible, in order not to state these responses loosely from general
impressions but in quantitative terms. ‘The principal criteria of
planarian reactions to light used in this study, follow:

1 Rate of Locomotion. Since the entire range of possible rates
of locomotion depends upon the structure of the worm and is not
very great, slight differences become significant.

2. Amountand Character of T urning, that is, whether persistent
or irregular, decided or vague, clockwise or contra-clockwise.

3. Change of Course. A change in the character, but not neces-
sarily in the direction, of the course is referred to here. “Circus
movements,” for example, would not be included under this head-
ing because the curving path in such cases, although constantly
changing in direction, does not change in character. “Tangents to
a circle, however, as well as angular and abrupt deviations from
a straight line may properly be regarded as changes of course.

4. Interval of Response. Vhe apparent effect of light is not
immediate in all cases, therefore, the time elapsing between the
application of the stimulus and the response to it is a valuable
measure of reaction.

5 Degree of Wandering. Ina sense the degree of wandering
shown by a worm is a measure of its indifference to the stimuli
acting upon it. It must be noted, however, that apparent indif-

The Reactions of Planarians to Light 49

ference may sometimes be due to a balance of opposing stimuli, in
which case wandering or aimlessness is not a true measure of the
effect of any single stimulus.

6 Orientation. his is a measure of behavior with reference
to the source of the light. It is expressed by the degree of posi-
tiveness or negativeness which the worm exhibits.

7 Duration of Movement. The time it takes a worm to tire
out when subjected to certain stimuli or, in other words, a measure
of fatigue.

8 Effect of Repetition. A measure of response is here referred
to which may be expressed quantitatively in units of time or quali-
tatively in manner of behavior.

g Wigwag Movements. These are waving movements of the
anterior end of the planarian, which appear to be a definite attempt
on the part of the worm to become adjusted to the stimuli acting
upon it.

10 The Time Required to Leave a Unit Circle. This is a
rather unsatisfactory criterion because it may indicate in some
cases a combination of several conditions as, for instance, latency
of response, rate of locomotion and degree of wandering.

11 Manner of Coming to Rest. Included under this heading
are such points as the position assumed, the locality selected, and
the abruptness of the act.

Naturally some of the foregoing measures of behavior will be
seen to have more application and value than others in the follow-
ing study.

V OBSERVATIONS
I PHOTOKINESIS

The term photokinesis was introduced by Engelmann (’83) to
denote the activities which are induced solely by the intensity of
light when the directive or orienting factor has been eliminated.

In this section will be considered, (A) the behavior of planarians
in the absence of light; (B) their behavior in different intensities
of non-directive light, and (C) the effect of abrupt changes, both
in time and space, in the intensity of non-directive light.

50 Herbert Eugene Walter

A Behavior in Dark

Darkness may be called the zero point in the scale of light inten-
sities. ‘That light is not essential to the activity of planarians is
shown by their performances in its absence, as is demonstrated
by the following facts.

Rate of Locomotion. ‘The average rate of ten individuals of
Planaria gonocephala was found to be 0.50 mm. per second in the
dark while the same ten worms, subjected to a light from above of
38 c.m.,? with all the other conditions unchanged, averaged 0.82
mm. per second.

Again, ten worms of the same species were allowed to travel in
the dark ten minutes in one set of experiments and six minutes in
another, when their average rates were found to be 0.42 and 0.57
mm. per second, respectively.

The method devised for obtaining the above records, previously
used in experiments upon fresh water snails (Walter, ’06), although
tedious was comparatively accurate. A clean glass plate was
submerged in a dish of water and the latter placed in a light-proof
receptacle. A single worm was then allowed to travel on this
glass for a unit of time, after which the plate was removed and
“developed” bk pouring over it powdered carmine shaken up in
water. A sufficier.t number of the insoluble carmine particles
adhered to the mucus-track left on the glass by the gliding worm
to make it possible to wipe dry the reverse side of the plate and
to trace thereon in ink the exact course taken by the worm. ‘This
permanent ink line was then measured by means of a map meas-
urer such as is in common use for measuring sinuous lines.

A series of experiments, to be described more in detail later
(Table III, p. 57), forms a basis of comparison with the foregoing
records in the dark, and further shows that there is an increase in
the rate of locomotion in the light.

Ten worms, subjected to various intensities of light projected
from above and ranging from less than one to several hundred
candle meters, showed rates which in all cases were greater than
the rate traveled in the dark.

$ The abbreviation c.m. is used to denote candle meters.

The Reactions of Planarians to Light 51

Turning. That planarians do more turning in the dark than
they do in various intensities of non-directive light is apparent from
the following table of percentages.

TABLE I
Per cent of LS of Planaria gonocephala in the dark and in various intensities uf use
——— =
Light in candle meters...........-..+ ° .. AI Ir | 39 | i 78 | eel 155 | 217 431 |Av. of all
(dark) | | | intensities
Percent off turmin pete -ater(eieistesinteeciet= | 87 76 | 66 | 69 | 81 | 67 | 75 | 77 | 65 72
. |
Per cent of straight paths............ | 13 24 | 34 | 31 | 19 | 33 | 25 | 23) 35 28
Number of observations............. 71 79 | 67 | 85 | 57 | 62 | 67 | 57 8

Furthermore, out of a total of 40 cases of turnings made by dif-
ferent individuals of Planaria gonocephala in the dark 23 were
clockwise and 23 contra-clockwise. This perfect balance in be-
havior did not recur when the same worms performed turning
evolutions in the light.

Change of Course. As to what constitutes “definite” and what
“indefinite”? changes of path, an S-shaped course is to be regarded
as an indefinite efenless wandering, whereas angles in a snare:
path or tangents in a curving path are classed as deanite responses
because they are what would normally occur if some directive stim-
ulus were interposed. It was found that P. gonocephala made
indefinite changes in its course more frequently in the dark than
in any series of light intensities to which it was subjected for an
equal length of time. On the other hand definite changes occurred
oftener in the light, although the factor of directive light had been
excluded.

Table II summarizes 350 recouas on 10 different worms with the
results reduced to percentages.

It will be seen that the percent of S-shaped (‘‘indefinite”’) paths
in the dark decidedly eclipses that which was made in any inten-
sity of light, while the per cent of angular and tangential paths
(“‘definite’’) laid in the dark is exceeded in every instance by that
made in any intensity of light with one exception, viz: II ¢.m.,
which, however, is not sufficient to change the average result.

Summary. Planarians move about in the dark but at a slower
rate than in non-directive light whatever the intensity. They

52 Herbert Eugene Walter

turn more in the dark than in the light, going clockwise or contra-
clockwise with equal readiness. Finally, they make more indef-
nite changes in their paths in the dark, but fewer definite changes
than in the light.

TABLE II

Percentage of definite and indefinite changes in the character of the course in dark and in light of different

intensities

Details of the several intensities employed

| Average |

Light in candle meters................-- o | for allin- Jo.94) 11 | 37 | 78 | 126) 155] 217] 431
|(dark)) tensities

Definite changes (angular or tangential

changes) percents ccoeecasssasanasn| 18 28.5 30°] 15) | 27 |) 32: |i 35;) 21 || 32" |'40
Indefinite changes (S-shaped paths), per) |

CONE serretays ee etic este tte stete pein esets 47 | 23 21 | 30 | 23 | 20 | 26 | 34 | 20 | 99
No change in character of course, percent} 35 | 48.5 | 49 | 54 | 50] 48 | 39 | 45 | 48 | 51

Number of observations......../.:..... 34 316 49 | 35 | 48 | 37 | 31 | 48 | 35 | 33

B Non-Drrective Light

a Apparatus

To test the effect of purely non-directive light, it is of course
necessary to eliminate the possible influence of directive light.
This may be done by projecting the light upon the moving worms
in such a way that they are unable to go either toward or away
from the source of the light. Whatever effect is obtained under
such circumstances must be ascribed to the non-directive power
of light.

The elimination of the directive influence of light can be accom-
plished by means of various devices. (1) The light may be made
to fall vertically from above upon a horizontal field; (2) it may be
reflected vertically from below so as to pass through a transparent
held at right angles to the plane of the field; (3) methods 1 and
2 may be combined. ‘The apparatus finally used in the majority
of experiments with non-directive light, was based upon the method
first mentioned.

The Reactions of Planarians to Light 53

Fig. 1 shows a diagrammatic vertical section of this apparatus.
The light (4), an incandescent electric lamp, was mounted in a
black sheet-iron hood (B) to prevent the escape of any lateral
light. This hood was suspended from the ceiling of the dark
room where the experiments were carried on and was arranged so
that it could be easily raised or lowered, thus changing the height
and consequently the intensity of the light with reference to any
fixed point below. In the hood, beneath the light, was supported

Fig.1 4, light; B, walls of hood; C, heat screen; DD, diaphragm; E, roof of hood; F, plate-glass
floor of aquarium; G, paraffine wall of aquarium; HH, diaphragm to cut off light reflections from
paraffine wall; J, wall of reflector box; ¥, open side of reflector box; K, mirror; L, walls of tunnel;
MM, black draperies; N, table.

a flat-bottomed, clear-glass dish (C) containing distilled water to
a depth of about three centimeters. The heat screenthus obtained
effectually filtered out the heat rays, allowing only the light rays
to pass through. A few inches under the heat screen was inserted
a diaphragm (D), painted black, the purpose of which was to aid
in cutting out side reflections besides allowing only a central col-
umn of light to escape below. A black sheet-iron roof (£) con-

54 Herbert Eugene Walter

fined the upward rays to reflections within the hood itself, at the
same time permitting the escape of heated air. Ona table directly
under the suspended light lay a horizontal sheet of plate glass
(F,) affixed to the upper surface of which was a circular ring
(G) made of a mixture of parafine and lampblack. There was
thus formed a circular water-tight aquarium twenty centimeters
in diameter and two centimeters deep, in which the worms could
be observed. On the top of this circular ring rested a black dia-
phragm (/7), the aperture of which was sufficiently small to exclude
any side reflections which might come from the black paraffine
wall.

The aquarium, it must be explained, did not rest directly on the
table but was mounted as the cover of a box (J), the interior of
which had been rendered largely free from reflecting surfaces by
the use of black camera-paint. One side of the box was removed
and, facing the opening thus made, a mirror (K) was placed at an
inclination of 45° with the horizon. The end of a square tun-
nel (L), ten feet long and made of black cloth stretched upon a
framework of wood, fitted close up to this opening. Suspended
from the lower edge of the hood and surrounding the aquarium
were adjustable black draperies (/) designed to shut out possible
side light and at the same time to allow a hole for the eye of the
observer. It will be seen that all light reaching the aquarium
comes from the lamp above by passing through the heat screen.

After illuminating the field of observation the light passes
through the glass floor of the aquarium and is reflected by the
mirror into the black tunnel. Most of the light is absorbed in the
tunnel, only an insignificant minimum being reflected back to the
aquarium floor. Otherwise complications in the character and
intensity of the light might arise.

By moving the hood (B) up and down and by using lamps of
different candle powers a variety of intensities was obtained. The
lamps used were tested by means of a Lummer-Brodhun pho-
tometer, the loss by reflection from the surface of the water both at
the heat screen and at the aquarium being reckoned out in deter-
mining the different intensities employed.

By simple observation, data for such criteria of behavior as

The Reactions of Planarians to Light 55

amount of turning, changes in course, degree of wandering, inter-
val of response and manner of coming to rest, could be obtained
in this apparatus with approximate correctness. To determine
the rate of locomotion, however, required a device which would
measure accurately the distance traveled in a unit of time. The

Fig.2 ABCF, pantograph; C, fixed point; D, paraffine wall of aquarium; £, plate glass bottom
of aquarium; F, place where the arm 4 is grasped by the operator. A style is located at end of
arm A, in contact with under side of aquariumfloor. J, style at end of tracing arm B, in contact with
smoked paper; , beginning of a course traced on the smoked paper; K, drawing board for attachment
of smoked paper; L, sheet of smoked paper fastened to drawing board; M, actual course of the worm.

method already mentioned of measuring rate from mucus-tracks
developed by means of powdered carmine, proved too tedious and
uncertain except for the worm’s maneuvers in the dark, when it
seemed the only available way.

56 Herbert Eugene Walter

To avoid the inconveniences of this method an attachment was
devised for directly duplicating the path of a worm by means of
a style traveling over a sheet of smoked paper. The records thus
traced were made permanent by immersing the smoked sheets in
a weak solution of resin in alcohol and allowing them to dry, after
which the paths could be accurately measured and the rates com-
puted.

The arrangement of this attachment, as seen from above, is
shown in Fig. 2. The diaphragm (Fig. 1, H) has been removed
for the sake of clearness. At the tip of arm 4F a style directed
upward comes in contact with the under surface of the aquarium
bottom (Fig. 1, F), while at the tip of arm B a similar style that
is pointed downward traces a line on the sheet of smoked paper L at
the left. After a little practice it was not difficult to keep the style
of arm 4 directly under the posterior end of a gliding worm, thus
duplicating its movements with considerable accuracy. The
expiration of any time interval can be indicated on the smoked
paper record by a crosswise scratch in the path.

Arm 4 was rendered as non-reflecting as possible by black cam-
era paint as well as by being made triangular in cross section with
the apex of the triangle upward. ‘Thus whatever rays struck it
from above were mostly either absorbed or reflected in a horizon-
tal direction, so that they did not reach the worm under experiment.

b- Results

Rate of Locomotion. Planaria gonocephala moves somewhat
more quickly in non-directive light than it does in dark. Ten
apparently normal and representative worms were selected and
isolated in individual aquaria. They were kept in the dim light
of the dark room in water of the same temperature as that of the
experimental aquarium in which they were observed. At the end
of thirty-four days of experimentation these worms showed prac-
tically the same average rates under the same intensities of light
as they did at first. By alternating the individuals these trials
were so made that fatigue effects had little part in the results,
while the succession of light intensities was varied in such a way

The Reactions of Planarians to Light 57

that cumulative effects and the influence of previous exposures
were largely avoided.
The results obtained in 259 trials are condensed in Table III.

TABLE III
Rate of locomotion in millimeters per second of Planaria gonocephala in various intensities of non-directive
light
| ie | a ‘ | =p |
Candlejmeterssaacacie sere see | ° | 0.94 | II | 39 | 78 | 126 | 155 217 | 431
Average mm. per sec....... 0.57 | 0.66 | 0.69 | 0.75 | 0.64 | 0.66 | 0.69 | 0.70 | 0.63
Number of records ............ | 30 «|. 28 30 | 29 | 27 || 301) 030 2 28

The mechanical stimulus resulting from the removal of the
worms, by means of a camel-hair brush, from their individual
aquaria to the observation aquarium was practically the same in
all cases as were al] the other external stimuli except light. The
difference in the rate of locomotion appearing in these averages
is, therefore, clearly due to differences in the light intensity em-
ployed.

It will be seen also that rate does not increase progressively with
intensity. ‘The series of rates and intensities under Table III,
if plotted in a frequency curve would give two modes, one at 39
and the other at 217 candle meters, with a slight depression be-
tween the two. Still, as has been already pointed out, any inten-
sity of light gives a faster rate than no light at all.

The slowest average rate was made under the highest intensity
of light employed. Certain facts to be brought forward later
favor the opinion that this was not an accidental result.

Under continuous exposure to one intensity of light the rate of
locomotion decreases. ‘The worms seem to “run down” gradually,
so that at the end of ten minutes their rate is only about half that
during the first minute. Data illustrating this point are given in
Table IV.

The rate of locomotion depends not so much upon the intensity
of light as upon other factors which tend to produce individual
behavior upon the part of each particular worm. Stated in another
way, there is greater variation between different individuals in the
average rate of their locomotion under all intensities than there

58 Herbert Eugene Walter

is in the average rate of all individuals collectively under different
intensities. [he data for this latter point based upon the average
rate of ten worms (259 observations) under different intensities
has already been given in Table III (p. 57). The extremes in
rate there shown are 0.57 mm. per sec. at zero intensity and 0.75

TABLE IV

Average rate of locomotion of Planaria gonocephala in successive minutes of exposure to 39 c.m. of non-
directive light

Number of minute.......) Ist | 2d | 3d | 4th sth | 6th | 7th 8th | gth | roth | 11th | 12th
| | | |

| | | | | |

| 4 > |b 48 0] Siena aes ee
3 29 | .25 | 29

No. of records averaged. . 17 | 15 | 12 | 7| 5 2

Rate in mm. per second..) .63 | .625) .565) .55 | -53 | -55 | -375] -39 | -39

mm. per sec. at 39 c.m. intensity, which makes a range of 0.18 mm.
per sec. When the same data are rearranged to show the average
rate for each individual for all intensities, as in Table V, the
extremes are 0.49 mm. per sec. and 0.83 mm. per sec. with a range
of 0.34 mm. per sec.

In fact the individual behavior of these ten worms, despite their
apparent similarity, was sufficiently distinct to allow each one to
be thereby identified.

Turning. Attention has already been called to the fact that
there is less turning in light of various intensities than in the dark.
A return to Table I will make plain that there fails to be any

TABLE V

Average rate of locomotion for each of ten worms (Planaria gonocephala) based on trials with non-directive

light of various intensities

Identification number of worm........ | I 2 3 4 5 6 7 8 9 10

pressed in mm. persec.............|0.79 |0-57 0.68 (0.64 0.83 |0.70 |o.72 0.58 [0-49 |0.62
! = ae Seas

definite correlation between the degree of intensity of the light and
the amount of turning, although the least turning occurs under
the highest intensity. This latter point, however, rests upon a
very slight difference and may not be significant. It 1s neverthe-
less worth mentioning, since it is in line with the effect of the

The Reactions of Planarians to Light 59

highest intensity upon rate, as well as with certain other evidence
to be discussed later.

The small excess of clockwise over contra-clockwise turnings
is not explainable upon the ground of varying intensities of light.
A distribution of the cases under the several intensities of light
(Table VI) makes it plain that this peculiarity is due rather to
individual causes than to light intensities. Indeed it would be
difhcult to conceive theoretically how varying intensities of non-
directive light could influence a worm in such a way as to affect
the direction in which it turns. ‘The natural expectation accord-
ing to chance would be an equal number of turnings in either
direction. ‘The excess of clockwise turns seems, therefore, un-
doubtedly due to internal causes which render certain worms
more liable to go one way than another. In fact, when the
records were arranged according to individual behavior it was
found that of the ten worms seven averaged a majority of clock-
wise turns while only three fell in the contra-clockwise column.

TABLE VI
Character of turning of Planaria gonocephala in non-directive light of various intensities
= 7 = ea =n “Sea se aiihea a = | an .
Light in candle meters...............| © | 0.94] II 39 | 78 126 | 155 | 217 | 431 |Total
Clockwise turns........... deemuaayie 2g bas. Wier mae | 17) x7) |22 | 24 | 22 | 203
Contra-clockwise turns............--| 23 23) ||) 37; 20 | 17 14 | 166

18 17 | 17

occur in the light than in the dark, but fewer “indefinite” changes.
This point requires no further exposition as its corollary has already
been given.

The behavior of the worm in this respect seems to be more
closely correlated with the highest intensity (431 c.m.) than with
any other. In the highest intensity employed there are indicated
(Table II, p. 52) 40 per cent of definite changes, which is con-
siderably in excess of the percentage of such changes made in any
other intensity. Onthe other hand indefinite, or S-shaped, changes
constitute only g per cent of all records taken at the highest
intensity, which is less than half the number of indefinite paths
made in any other intensity.

60 Herbert Eugene Walter

While the extremes of the series of definite changes indicate a
general rise in the percentage of their occurrence with an increase
of intensity, and while in the same way the extremes of the series
of indefinite changes suggest in general a decrease of frequency
with the increase of intensity, it can hardly be maintained that
the character of the changes in course is definitely correlated in
the majority of cases with changes in intensity.

Degree of Wandering. Wandering is not closely correlated
with the intensities of light. In Table VII, which deals with the
percentage of straight paths made by P. gonocephala under dif-
ferent intensities of non-directive light, this fact is expressed nega-
tively, since it is held that a straight path is a good indication of
the absence of aimlessness or wandering and may thus serve as a
negative measure of such behavior.

TABLE VII

Percentage of straight paths made by P. gonocephala in the dark and also in non-directive light of different

intensities

Light in candle meters... . Hogeoanmnsite o |0.94| 11 | 39

In this respect again the behavior of the worms under the high-
est intensity 1s more pronounced than under any other intensity
since the greatest number of straight paths were laid at an inten-
sity of 431 c.m.

Interval of Response. There seems to be some evidence that
the interval of time elapsing between the reception of a light stim-
ulus on the part of a worm and its consequent response, may be
quite considerable. ‘Three facts were established that may sup-
port this conclusion.

First, when two-minute records were made under various inten-
sities, it was found that the worms averaged a faster rate during
the second minute of exposure to the light than during the first,
in spite of the facts that the mechanical stimulus due to placing
the worm in the light machine had a more quickening influence
during the first minute and that the fatigue effects were more
likely to appear during the second minute. The actual figures

The Reactions of Planarians to Light 61

for the above statement, based upon 240 two-minute trials under
various intensities, are 0.645 mm. per sec., the average during the
first minute, as against 0.713 mm. per sec., the average during
the second minute.

Secondly, in these 240 trials, the percentage of turning under all
intensities 1s greater during the first minute than during the sec-
ond, being 87 per cent and 57 per cent, respectively. “This result
may possibly be conceived to be due to a greater steadying influ-
ence of the light during the second minute than during the first
and to a consequent greater turning than during the first minute.
But on the other hand a similar decrease of turning, although not
so pronounced, took place during the second minute when the
worms were in the dark. It must be admitted, therefore, that the
fact of less turning during the second minute may have nothing
to do with the interval of response.

Thirdly, on several occasions a notable piece of behavior was
observed, which may have a bearing on the interval of response.
The phenomenon in question AGars occurred in connection with
a modification of the experimental held within the light machine
to be more fully described later. Briefly this modification con-
sisted in making a field of two distinct intensities of light, the
latter being projected vertically from above in such a way that a
sharp line of demarkation formed a boundary between the two
areas. Ordinarily when the worms reached this boundary line
as they glided from one intensity to another, they responded
promptly to the stimulus caused by the change of intensity. Sev-
eral times, however, they were observed to travel indifferently
exactly along this dividing line for a distance of several centimeters
with half the body in one intensity and half in the other. This
curious fact lends itself to various interpretations, one of which
is that the response to a new intensity may not be, in all cases,
immediate.

Manner of Coming to Rest. During the experiments made
in the non-directive light apparatus previously described, nor-
mal worms could never be induced to come to rest in the light.
If allowed to remain in the aquarium they would wander about
until they reached the shadow under the diaphragm (Fig. 1, H),

62 Herbert Eugene Walter

where they finally stopped, usually in the angle formed by the
parafhne wall and the bottom.

Loeb’s conclusion (’93b, p. 101) that planarians subjected to
directive light come to rest in regions of least intensity, seems
therefore to be equally true of planarians in non-directive light.

Summary. In non-directive light Planaria gonocephala moves
faster, turns less and makes more “‘ definite’ but fewer ‘‘indefinite”’
changes than in the dark. Rate of locomotion; amount of turning;
changes in the character of the course, as well as the amount of
wandering, do not appear to be correlated with varying light inten-
sities, unless in the following instance. Under the highest inten-
sity employed, namely, 431 c.m., occurred the slowest rate; the
least turning; the greatest number of “definite” and the fewest
“indefinite”? responses, together with the straightest paths. The
excess of clockwise over contra-clockwise turnings throughout the
series of intensities is probably not attributable to light.

Continuous exposure to light results in a decreasing rate of loco-
motion, although in the second minute of movement as compared
with the first an increase in the rate of locomotion takes place,
while fewer turnings occur.

Rate of locomotion is less influenced by differences in light inten-
sity than by certain internal factors which go to make up what
may be termed the individuality of different worms. Individ-
ual worms may sometimes fail to respond for a considerable inter-
val of time to light stimuli that ordinarily produce immediate
effects.

Finally, planarians subjected to non-directive light come to
rest in regions of lessened light intensity the same as they do in
directive light.

“Gi Abrupt Changes in Intensity

Abrupt changes in intensity may be of two kinds: either with
reference principally to time or to space. First, those changes are
abrupt zm time in which light or dark is suddenly thrown upon the
worm, and secondly, those changes are abrupt 1m space in which
a moving worm passes immediately from an area of one intensity
into a sharply defined area of a different intensity. ‘This topic

The Reactions of Planarians to Light 63

will be discussed here only in its relation to non-directive light, the
effects of sudden changes in directive light coming more properly
in a later section.

a Abrupt Changes of Light Intensity in Time

Whenever worms were left over night in the experimental aqua-
rium completely shut off from light, a large proportion of them
would be found at rest in the morning when the light in the hood
was again turned on. By removing ie diaphragm (Fig. 1, H),
under mie edge of which near the penaiine wall the worms were
usually enllcceces it was possible without any mechanical disturb-
ance to subject resting worms to sudden non-directive light after
a prolonged period of complete darkness. This sudden stim-
ulus rarely had an instantaneous effect. ‘The interval of response
was often several minutes and frequently non-directive light alone
proved insufficient to start the worms into activity.

No sudden increase of intensity ever proved powerful enough
to throw a gliding worm into the more rapid method of crawling.
Pearl (’03, p. 551) stated the same fact after subjecting planarians
to much stronger intensities of light than were employed in the
present experiments.

It was found that P. gonocephala showed a decided response—
either some change in course or a wigwag motion of the anterior
end—more frequently when suddenly subjected to dark than
to light. By inserting a key into the electric circuit it was
possible to control the light in the hood to a fraction of a second.
Worms in complete darkness were by this means subjected to
various intervals of sudden light and worms in light to intervals of
sudden dark, the results being at once noted. While the worms
were in the dark their behavior could not, of course, be directly
observed, but by watching them closely just before the light was
turned off and also the instant it was turned on again there was
no great difficulty in determining whether a response had occurred
during the interval. The results obtained from nearly a thousand
trials are indicated in Table VIII.

It will be seen from this table that there are more responses
than failures to respond and that the responses occur more fre-

64 Herbert Eugene Walter

quently when the worms are suddenly subjected to dark than to
light.

It may be further noted that the excess of the responses in the
dark over those in the light increases with the interval of exposure,
indicating that the worm’s adjustment to a change in the light
stimulus affecting it is not in all cases immediate.

The effect of previous exposure, whether to several hours of
dark or light, is a factor in these results which will be considered
more properly later on.

TABLE VIII

Percentage of the responses of P. gonocephala in various intervals of time when suddenly subjected to

dark and to light of 39 c.m.

Number of seconds exposed..............-- 5 | 10 15 20 25 30 | Average
Percentage of responses in light............ 51 | 59 54 54 48 46 52
Percentage of responses in dark............. 63 66 73 75 71 71 7°
Excess of responses in dark....... ROSA OS 12 7 19 21 23 25 18

It should be added that Bdelloura gives a remarkable response
when enveloped in sudden darkness. It will frequently forsake
its attachment under these circumstances and unattached in the
water go through violent contortions. ‘This striking response can
be called forth by an exceedingly brief interval of dark, namely,
the shortest time required to turn the electric light off and on.
Nagel (’94, p. 387) speaks of animals thus affected by sudden
shadow as “skioptic.”’

The relation of Bdelloura to light falls into a somewhat different
category, however, than that of the fresh-water planarians, since
Bdelloura is positive to light, while fresh-water flat-worms are
negative.

b Abrupt Changes of Light Intensity in Space

Several devices were employed to test the behavior of planarians
passing abruptly from an area of one intensity of non-directive
light into another. ‘The most successful device tried was that in
which two lights of different intensities were mounted overhead

The Reactions of Planarians to Light 65

in the hood of the apparatus already described in Fig. 1, the
mingling of their rays being prevented by the insertion of a ver-
tical diaphragm (Fig. 3, C), which extended from the region
between the lights down to the surface of the aquarium. In order
to place the diaphragm in position it was, of course, necessary to
remove the heat screen (Fig. 1, C), the presence or absence of
which, however, would not have affected the results sought since
the water in the aquarium itself was nearly 2 cm. deep and thus

Fig. 3. A, stationary light; B, sheet iron walls of hood; C, vertical diaphragm separating the two
lights; D, horizontal diaphragm; £, sheet iron roof of hood; F, plate glass aquarium floor; G, paraffine
wall of aquarium; HH, diaphragm to shut off reflections from wall of aquarium; J, wall of reflector box;
F, open side of box; K, mirror; L, black tunnel; M, black draperies cutting off side light; N, table sup-
porting reflector box and end of tunnel; 0, movable light; P, track for movable light; 9, narrow, hori-
zontal diaphragm attached at right angles tothe lower side of the diaphragm C, in order to prevent
the light rays from the two sources of light, 4 and O, from overlapping.

constituted an efhcient heat screen. By keeping the hood sta-
tionary and causing one of the lights (Fig. 3, O) to slide up and
down at will, it was possible to bring about various contrasts of

66 Herbert Eugene Walter

intensity in the field below. The complete plan of the appa-
ratus Is given in Fig. 3.

The principal variations in the behavior of Dendroccelum and
Phagocata upon reaching the critical line separating the areas of
two intensities are indicated diagrammatically in Fig. 4.

The dotted line represents the boundary separating two areas
of different light intensities. The arrows represent the types of
paths made by Dendroccelum and Phagocata. For the sake of
simplicity the worms are represented as going in one direction;
that is, into one of the two contrasting intensities, but the same
types of paths resulted as well when the opposite direction was
taken. The angles made in crossing the
critical line were also more varied than those
represented in the diagram.

Type 4 represents a passage without re-
sponse; B,an angular change of course made
at the critical line; C and F, aloop-like return
effected after a short excursion into the new
intensity, and G,a sharp turning aside, while
A indicates a halt at the critical line, as if a
barrier had been encountered. Finally D and
E represent a temporary pause on the part
of the worm accompanied by wigwag move-
ments of the anterior end of the body. In the
case of D the wigwagging is immediate, but
E typifies a case when there occurred in the
response an interval of such a nature that the

' significant movements were not made until
Fig. 4 the worm had advanced at least its own length
into the new area.

Of these types all, with the exception of 4, are to be regarded
as reactions to differences in intensity encountered. The most
questionable are the infrequent types C and F, which may be
otherwise explained as arcs in a curving course which might have
occurred in a field of uniform intensity. By far the commonest
type was D, plainly the least doubtful of the series.

The Reactions of Planarians to Light 67

As a result of over 3000 observations on the manner in which
the critical line separating the two intensities was passed, three
facts become evident. First, responses were considerably more

TABLE IX

Kind and percentage of responses of Dendrocalum and Phagocata in passing from one intensity of non-

directive light to another

Turn-backs|
No Wigwags and full Loops Angular Total
Character of course responses) (Types | stops | (Types courses reece
(Type 4) Dand E) | (Types | C and F)| (Type B)
G and H)
Going into greater intensity,
PETCEN Greys eteressfulshessreleat ests 79 II 6 2 2 2I
Going into lesser intensity,
PEM CODG gararaintatelstetar lererstaroks 5° 36 5 8 I 50
Average responses, per cent.| 64.5 tals 5-5 | 5.0 Aly 35°5

frequent when the worms were passing into the lesser intensity
than they were when entering the greater intensity. Secondly,
lack of response is more frequent than a visible response of
any kind since 64.5 per cent of the crossings made over the
critical line were of the type 4. Thirdly, the responses at the
critical line were more frequent when the worm was upside down,
1. e.. moving on the surface film, than when it was on the floor
of the aquarium. This latter point was illustrated most fully
by Phagocata, which, being an active worm, takes quite readily
to the surface film, so that it was possible with this species to get
a series of observations in which the behavior when crossing the
critical line on the bottom of the aquarium could be compared
with that when the same line was encountered at the surface film.
Table X contains the results of these observations.

The doubling of responses when the worm is on the surface
film is probably not due to an unequal receptivity of light stimulus
by the dorsal and ventral surfaces of the planarian as might at
first thought seem possible. As will be shown further on, the
worm’s rate of locomotion on the bottom of the aquarium is nearly
the same whether the light comes from below or from above, pro-

68 Herbert Eugene Walter

vided the amount of light in both cases is equal. Planarians, as
Pearl has emphasized, are strongly thigmotactic. Naturally, then,
their response to contact is much greater when they are on the
glass bottom of the aquarium than when they are suspended on
the less resistant surface film. In other words, the less the worm
is influenced by the stimulus of contact the freer it is to respond
to the stimulus of light.

TABLE X

Percentage of the responses made by Phagocata at the critical line separating two intensities of non-direc-

tive light either on the bottom of the aquarium or on the surface film

| Number of No response Response
| observations per cent per cent
Ounithe surfacedilia!acones chs apeanastosuiss cess 740 45% 544
On: thetbottome. oancrsosen ee eee aneiacg cee ai 1664 76 24
MMotalisiepsannc te oxelateernvctsy se corers/o\sralsinyo: Se co-fiassiecto 2404 60} 39%

Finally, a series of experiments was tried in which the contrast
between two intensities was varied by raising or lowering one of
the lights in the hood. It was found that the responses made by
Phagocata under these circumstances increased with the increase
in contrast between the two intensities as shown on the bottom
line of Table XI, where these contrasting intensities are expressed
in a ratio between the constant light taken as unity and the movy-
able light.

The fact that responses by no means invariably occur when
bright light and complete darkness are suddenly substituted for
each other (see Table VIII) rendered a further extension of this
series unnecessary. The contrasts here used form probably a
much greater range of intensity contrasts than the worms ever
encounter in nature.

Attention to the details presented in Table XI brings to light
the fact that, although the number of responses is correlated in a
general way with an increase in the contrast between the two
illuminated areas, as shown in the bottom line of the table, yet
the percentage of the responses is further influenced by the actual
degree of the intensities employed. For example, when the two

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IX ATAVL

70 Herbert Eugene Walter

areas of light were respectively 33.16 and 68.18 c.m. the ratio was
practically the same as when the two intensities were 16.3 and
33-16 c.m., yet the percentage of responses in the two cases is
decidedly different, being 10.5 per cent in the former, and 56 per
cent in the latter case W hen ve lesser of the two lights was 33.16
c.m. there were invariably fewer responses than aa the lesser
light was 16.3 c.m. The latter intensity is undoubtedly nearer
the planarian’s optimum intensity, and the apparently inhibitive
action of the higher intensities agrees perfectly with certain facts
already detailed, as, for instance, that the activities of Planaria
gonocephala were less pronounced at 431 c.m. than in lesser inten-
sities; and, again, that all planarians show more responses on
going into a lesser than when going into a greater intensity.

Attempts were made in some other ways to subject planarians
to areas of contrasted intensities and, although the results were
less satisfactory in general than those obtained by the method of
using two buerhead lights of different intensities just described,
yet certain facts were ibeatie out that may be worth recording.

In the first of these attempts two concentric rings of white
paper, each about two centimeters wide and having between them
a space of a couple of centimeters, were fastened to the under sur-
face of the aquarium floor. The white paper thus arranged
reflected the light upward and made areas of gradually increased
intensity as compared with the remainder of the aquarium floor
through which the light passed without reflection. Worms placed
in the center of these circles would consequently be obliged to
pass from one intensity of light directly to another, ee er the
direction of the radius they might be taking. When worms were
actually tested, it was found that they exhibited considerable
modification in their movements, particularly when approaching
the edge of the paper backgrounds.

Owing to the considerable thickness of the plate-glass floor of
the aquarium as well as to the fact that white paper is a surface
which scatters the light falling upon it, it was found that there was
formed, not a sharp line of demarkation between two intensities,
but rather a penumbra-like margin of intermediate light. This
apparatus was therefore abandoned as unsatisfactory.

The Reactions of Planarians to Light 71

The difficulties presented by paper as a reflecting surface largely
disappeared by the substitution of a plain mirror in its place, since
the surface of a mirror is such that all the light striking it at right
angles is reflected at right angles. When, therefore, an unmounted
mirror was brought into contact with half of the under surface of
the aquarium floor the whole field was thereby divided into two
regions sharply separated from each other. Of these one was
supplied with light from above only, while the other received the
same light plus Aeanl: an equal amount reflected from the mirror
below. With the aid of this device an increase of II per cent was
gained over the responses obtained when white paper instead of
a mirror was used as a reflector. Both Phagocata and Dendro-
ccelum were tried by this method. In 76 per cent of the trials
made, 7. ¢., in 125 cases out of 165, the worms showed a visible
modification in their behavior on reaching the boundary of the
two areas of light. It was nevertheless decided that this method
was an uncertain test of behavior, since the body of the worm,
although fairly translucent, would by no means allow all the light
that fell upon it to pass through and be reflected, and consequently
the difference of the two intensities to which it was being sub-
jected could not be easily estimated.

Summary. When sudden light or dark envelops planarians
(Dendroccelum, Phagocata and Planaria) the response, if any
occurs, is often not immediate.

No one of the intensities of light which were employed in these
experiments when introduced suddenly was sufficient to make the
worms forsake gliding for crawling.

Sudden dark calls out more responses than sudden light, while
the number of responses increases with an increasing interval of
exposure to the stimulus. Bdelloura is decidedly “‘skioptic.”’

Worms encountering the edge of a reflecting area which increases
the intensity of the light without introducing any other barrier,
show a marked degree of response. ‘The percentage of response
is considerably larger when a mirror instead of white paper is used
to produce the reflecting surface. If worms are allowed to pass
from one intensity to another sharply separated from it, their
responses are more frequent upon passing into the lesser intensity

72 Herbeit Eugene Walter

than when going into the greater. The average number of fail-
ures to respond to these contrasts of intensity reaches about two
out of three.

Phagocata, at the critical line separating two contrasting inten-
sities, responds oftener when on the surface film than when glid-
ing over the bottom of the aquarium.

The number of responses increases with the increase in the con-
trast between the two intensities employ ed, but the percentage of
response is greater, regardless of ratio, when one of the lights is of
low intensity (13.6 c.m.) than when both are of higher intensity

(33 + c.m.)
2 PHOTOTAXIS

The term “phototaxis” was introduced by Strasburger (’78) in
a study of certain swarm-spores, to indicate movements which
were parallel with incident light rays. The term has since been
extended by several authors to include similar movements on the
part of animals. Any organism is said to be positively phototac-
tic when it moves toward the source of light in the direction of the
rays and negatively phototactic when it goes in the opposite direc-
tion.

The purpose of this section is to consider the phototactic move-
ments of planarians, as distinct from their photokinetic behavior,
(A) when the light remains constant, (B) when the light is changed
either (a) in intensity or (b) in direction, and (C) when phototaxis
is combined with responses of a different kind.

A In Constant Directive Light

Orientation. With the exception of Bdelloura all the planarians
studied are, under normal conditions, negatively phototactic so
far as their first movements in directive light are concerned. To
obtain quantitative data for this statement it was necessary to
construct an apparatus in which the worms to be tested could be
placed quickly and with as little mechanical disturbance as possible
in the center of a unit circle with the long axis at right angles to the
direction of incident light. The circle was marked off into degrees
so that by noting the place at which a worm made its exit a quan-

The Reactions of Planarians to Light 73

titative measure of the amount of turning toward or away from
the source of the light under the given conditions was obtained.

The apparatus finally utilized for this experiment was based
upon a device employed by Parker and Burnett (’oo) in testing
the relative behavior of normal and eyeless planarians when sub-
jected to directive light. Its arrangement is shown in Fig. 5.

On the top of a table (4) in the dark room was placed a rectan-
gular aquarium (BCDE), the ends of which (BE and CD) were

Fig. 5 A, Top of table; BCDE, rectangular aquarium; BE, glass end; FG, round swinging
aquarium; H, copper wire attached to ceiling and supporting the swinging aquarium FG; J, movable
light; 7, diaphragm; K, surface of water in outer aquarium; L, surface of water in inner aquarium;
M, lens.

made of glass while the floor and sides were of wood painted with
camera-black. Within this aquarium a second cylindrical one
(FG), made entirely of thin glass and measuring 20 cm. in diam-
eter, was suspended from the ceiling by means of a fine wire (/7)
attached to a swivel to allow turning. On the floor of the outer
aquarium and directly beneath the inner one was drawn a circle

74 Herbert Eugene Walter

10 cm. in diameter and marked off plainly into arcs measuring
5 degrees each. An incandescent lamp (/), placed on the
table at approximately the height of the inner aquarium floor,
could be manipulated at any desired distance, while a diaphragm
(Ff) prevented much of the light from reaching either the upper
surface of the water contained in the two vessels or the floor of the
outer aquarium whence it would be reflected. A biconvex lens was
then so interposed as to make the light rays practically parallel
upon theiremergence fromit. ‘Their course through the inner aqua-
rium was kept parallel by means of the medium of water on both
its inner and outer sides. A nearly uniform intensity over the
entire floor of the swinging aquarium was thus obtained and the
objection arising when the inner aquarium is used in air, viz: that
it acts as a converging lens, was obviated. Side reflections were
eliminated by enclosing the light (/), together with the interven-
ing space between it and the diaphragm, with black screens.

When a worm introduced into the inner aquarium began to
glide, it could with slight mechanical disturbance be quickly cored)
by means of moving cen aquarium, into any desired posi-
tion with reference to the light, and then swung so as to bring its
posterior end exactly over the center of the stationary circle
below.

Various species of planarians were started in this manner at
right angles to the light. Out of 386 cases, 371, or 96 per cent,
emerged from the 10 cm. circle at a point farther away from the
light than that toward which they were originally directed. ‘This
is taken to mean that 96 times out of a hundred the worms were
negatively phototactic. If, however, the method of reckoning
negativeness employed by Parker and Arkin (’o1) on the earth-
worm is used, the foregoing per cent would be somewhat less.
These authors assume (’o1, p. 28) that the apparently positive
responses of a normally negative animal, such as the earthworm,
may be due to causes other than light, in which case an equal
number of responses of like nature might be expected to occur on
the negative side as well as on the positive. A number equal to
the sum of these apparently positive responses should therefore
be subtracted from the total of the apparently negative responses

The Reactions of Planarians to Light 75
in order to obtain approximately the amount of unquestionable
negativeness. By following this method in the case just given,
the per cent of negativeness would be g2 instead of 96, but since
this method assumes that normally negative worms are never posi-
tive, which is contrary to the evidence to be given later, the most
accurate estimate of negativeness would probably fall somewhere
between these two percentages.

Bdelloura, on the other hand, behaves in the same way only
three times out of ten, therefore showing itself to be positively
phototactic.

This difference in orientation becomes more marked if the total
number of degrees, that is, the amount of positiveness and nega-
tiveness of emergence from the circle is used as the basis of reckon-
ing, instead of only the number of times of emergence. Such a
quantitative computation is shown in Table XII.

TABLE XII

Amount and kind of orientation to directive light exhibited by various species of planarians in 396 trials

| Number of Total de- | Total de- |Percentage of Percentage of

trials grees positive |grees negative) degrees neg. | degrees pos.
Negative worms (Dendroce-
lum, Planaria, Phagocata). 386 | 566 10157 94.7 ces
Positive worms (Bdelloura) .. .| 10 397 50 11.2 88.8

Although the actual number of trials for Bdelloura in this table
is small, they are characteristic of what was observed in a large
number of unrecorded instances.

The amount a planarian may deviate from the direction in
which it is pointed, depends upon the direction of the light imping-
ing upon it. A negative species deviates from a straight course
least when headed away from the source of the light and most
when headed toward it, while an intermediate degree of deviation
occurs when the direction of the light is at right angles to the long
axis of the worm. In the case of Bdelloura the converse is true,
as shown in Table XIII.

Rate of Locomotion. In obtaining the rate of locomotion of
worms subjected to directive light, the double aquarium apparatus

76 Herbert Eugene Walter

just described was used. After the worm to be tested had been
placed in the inner aquarium and had begun gliding, it was so
oriented that the tip of its posterior end came precisely over the
center of the subjacent circle 10 cm. in diameter. The exact
time of its departure from the center of the circle was then noted
and the instant thereafter that the tip of the posterior end passed
over the circumference of the circle was again taken and the
worm’s course plotted at once on a duplicate circle sheet. Each
worm was given four trials in this manner, being started in four
different directions, toward the light, away from the light, and
with the long axis of the body at right angles to the light, first
with one side to the light and then with the other.

TABLE XIII
Amount of average deviation tn 2400 trials expressed in degrees of a circle, exhibited by negative planarians,
(Dendrocelum, Planaria and Phagocata), and a positive one (Bdelloura) when pointed toward,

away from, and at right angles to the source of light

Direction in which the worm was pointed with regard)

tOsthesl ser hiti tess /alejararacstcvatevaveracchey shia. ’eps/s a wierd ontynusith one At right angles Toward Away from
Negative planarians, degrees.........-.-+++++++s0-- 48.1 128.7 2759
Positive planarians, degrees..... : eveuarsvele Waxege 49. 39-3 132.1

The time of the worm’s emergence from the circle was not taken
with a stop-watch because the observer’s hands were otherwise
occupied. Instead a small clock, ticking half-seconds, was placed
conveniently near. By counting the number of ticks during the
interval of the worm’s transit from the center to the circumference
of the circle the time consumed could be determined within less
than a half-second. After tracing the worm’s course on a dupli-
cate circle sheet and measuring the same by means of a map
measurer, a unit of distance was obtained, which together with
the known unit of time consumed in covering this distance, fur-
nished all the data necessary for computing the rate of locomotion.

Ten representatives of Dendroccelum lacteum, Planaria macu-
lata, Phagocata gracilis and Planaria gonocephala respectively
were given four trials apiece by the method just explained. ‘The
results are presented in Table XIV. From the 160 records thus
obtained it becomes evident that the average rate of locomotion

The Reactions of Planarians to Light fh

is greatest when the worms are pointed toward the light, and least
when they are pointed in the opposite direction, while an inter-
mediate rate occurs when they are started at right angles to the
light.

This result is at variance with the findings of Parker and Bur-
nett (’00, p. 381), who incidentally reported that Planaria gono-
cephala when started away from the light traveled faster than
when started toward the light.

TABLE XIV

Average rate of locomotion, expressed in mm. per sec., of various species of planarians when started
torward, away from, and at right angles to the source of directive light of 27 c.m. intensity.

: Dendroceelum | Planaria |Phagocata Planaria Total
Species |

lacteum | maculata | gracilis | gonocephala| average

Direction in which the worm was

pointed with reference to the light

At right angles'.....5....2.% 0.855 1.475 1.445 0.980 1.19
LGR Eid logoseoopagnaouneder 0.g10 1.505 1.430 1.205 1.26
wma ye ir OM cra )axerereesey~layate/2)<yei2 0.795 | 1.440 1.310 1.090 1.16

It was further found that, regardless of the direction in which the
worms were started, there was a gradual decrease of the rate dur-
ing the four successive trials. “The order in which different worms
were oriented during the four trials was arranged so as to neutral-
ize the possible effect of the sequence in the direction started. In
Table XV the data for 200 trials are arranged to express this
slowing down of the rate.

TABLE XV

Average decrease in rate of locomotion for 50 planartans during four successive trials while subjected to

directive light of 27 c.m.

IN rina exo fi tari all sa sjoyefays)-\sh-00 2 abate ace nayoxste = lava'e) yafelen sie First | Second Third Fourth
i}
|

Averapexatenmimm.) per SeGer cieefeleiers ware laleistaiets 1.140 1.130 1.075 1.070

Various factors influencing the rate of locomotion, such as the
intensity of light, the size and species of the worm, the amount
of pigment present in the body and the general physiological state
of the animal under experimentation, will be more suitably dis-
cussed in other connections.

78 Herbert Eugene Walter

Change in Character of Course. When several specimens of
Phagocata were placed in a square aquarium which received light
solely from one side, their first movements were plainly negative,
that is, away from the light. After a brief interval, however, it
was seen that apparently as many worms were going toward the
light as in the opposite direction. In fact an actual count showed
that in a certain interval of time 43 worms passed a central point
going toward the light while 44 passed the same point in the oppo-
site direction. This apparent change in the character of the
course was probably due, not to any change in the degree of nega-
tiveness of the animal, but rather to the fact that the impulse to
keep moving in some direction is stronger than the impulse to neg-
ative phototaxis. Consequently when the limit of the aquarium
in a negative direction is reached a worm, since it normally travels
in straight lines or sweeping curves and does not turn around
and around in one spot, continues its locomotion in the direction
of least resistance, namely, back toward the light. It will be
remembered that Loeb (’93b) has called attention to this fact by
saying that planarians are not negatively “heliotropic”’ in a strict
sense because they do not remain as far away from the source of
light as they can get.

Among various observations made with other ends in view,
there were numerous incidental cases of a normally negative
worm making an unexpected positive response even from the first
moment of being subjected to the light stimulus. ‘This occasion]
positiveness is clearly apparent from the general fact already
noted that four times out of a hundred the average negative pla-
narian turns toward the light.

Two definite instances of a reversal in the character of response
may be cited.

The first was the case of a Phagocata in the double aquarium,
which became increasingly positive through twelve successive trials.
Its average emergence from the circle for the first four trials was
45°, which is a normal negative result, since go° represents com-
plete indifference. In the next four trials, however, the average
was 100°, that is, slightly positive, and in the last four, 124°,
which is decidedly positive, as shown diagrammatically in Fig. 6.

The Reactions of Planarians to Light 79

In the other instance an individual worm, Planaria gonocephala,
made the erratic average emergence from a circle of 145°, just
35° short of absolute positiveness. ‘This worm was carefully
isolated and tested again four days later under identical external
conditions when it was found to have returned to a normal nega-
tive condition by showing an average record of 56°.

Accuracy of Orientation. It was found to be frequently the case
that when negative worms were subjected to directive light their
first movement instead of being directly away from the source of
light formed a path in a diagonal direction. “This tendency to

rege GO

90° AS

Light 180° O°

90°
Fig. 6 The arrow at the left represents the constant direction of the light. In eachof the three sets
of trials each worm was headed successively toward 0°, the upper (in the diagram) 90°, 180°, and the
lower go°. The point of average emergence for the first set of trials—supposing the records of the
lower semicircle to have been transferred to the upper semicircle—was at 45°, of the second set,
at 100°, and of the third set, at 124°.

travel diagonally away from the light has also been noted in the
case of the earthworm by Smith (’02, p. 469).

If the negative phototaxis of planarians is to be explained on
the theory of tropisms, and if, moreover, the eyes, as Hesse (97)
maintains, are the principal organs which, when unequally illumi-
nated, cause the directive response, it may be shown that possi-
bly the arrangement of the crescentic pigment shields around the
sensory cells of the eyes is such that equal stimulation of both eyes
is just as certainly received by the worm when it is in a position
diagonal to the light as when it is pointed directly away from the

light.

80 Herbert Eugene Walter

By reference to Fig. 7, in which the relative size of the eyes 1s
somewhat exaggerated and made diagrammatic for sake of clear-
ness, it will be seen that no more light reaches the sensory cells of
either eye from position 4, the diagonal position, than from posi-
tion B, and that it is only when the light comes from some source
more lateral than 4 that the left eye receives more illumination
than the right.

This view may furnish a possible explanation of the diagonal
paths representing imperfect orientation among planarians, but it
can in nowise apply to the case of earthworms since in them direc-
tion eyes are absent.

Fig. 7 A, diagonal direction of light; B, posterior direction of light; C, location of sensory cells;

D, pigment shield.

Degree of Wandering. ‘The degree of wandering decreases with
an increase of intensity. It may be found approximately through
the degree of error in orientation in a unit space under different
intensities of light, for perfect orientation signifies the minimum
of random wandering and, conversely, the greater the error of
orientation the greater the probable wandering.

The error of orientation expressed in percentages was computed
as follows. With a negative worm emergence from the circle at

The Reactions of Planarians to Light SI

a point directly opposite the light was reckoned as 0 per cent of
error, whereas emergence at a point directly toward the light was
reckoned as 100 per cent, or a maximum of error in orientation.
The orientation value at these two extremes having been estab-
lished, the percentage of error which occurs when the worm
emerges at any intermediate position on the circumference of the
circle may be easily determined.

TABLE XVI

Average degree of error in orientation made by various species of planarians during 300 trials in directive
light of different intensities

| Percentage of error in orientation

iene 4 When started

Mea etarte! = away from the Average
ward the light light
3-3 candle meters......- poopdso ss OnoeandeDane 34 11.5 22.7
FLOIGALI GAG TTNCLENS = ans Vaperalsy/atece(ye/<tatsisraial svete ave4e <ns/e 32 | 12 22.0
BAL Cand lewn etersemrstar sate rsiateisieie/s aleleteteiateiefeinve -| 31 | 10 20.5
| ;
MAVCLA RE otatateletaieveyeteteCayetecerctetenesctcus crs| sobs she, ze1 32 II

From this table it appears that there is three times as much
wandering, or error of orientation, by worms headed toward the
light, as by those headed away from it. This doubtless indicates
that orientation is a more complicated process in the former case
than in the latter.

Duration of Activity. Superficial observation is sufficient to
establish the fact that different species of planarians when set into
activity in directive light show decided differences with regard to
the length of time they normally continue in motion before com-
ing to rest. Among the forms experimented upon, Bdelloura

came to a stand-still in light soonest and Phagocata latest.! Fatigue
in itself is by no means the inevitable result of continued activity
on the part of anorganism. For instance, Hodge and Aikens (95)
observed a Vorticella continuously for 36 hours, during which
time its regular ciliary and contractile movements continued unin-
terruptedly, while Radl (’or) found that the eye of Daphnia when

82 Herhert Eugene Walter

light was flashed upon it vibrated as vigorously after the experiment
had been repeated 410 times in close succession as it did at first.
An attempt was made with Planaria maculata to see how long
activity would continue in a succession of trials in directive light.
The worm was started on the middle of an aquarium floor and
allowed to glide in any direction. As soon as it stopped and
assumed the relaxed contour of the resting worm, the time required
for the journey being noted, it was immediately returned to the
starting point. Subjected to this treatment, the worm made 39
trips, which in general occupied an ever decreasing length of time,
ranging from 18 minutes to 1? minutes, or an average of 5 minutes
and 53 seconds each. When returned to the starting point the
fortieth time the worm refused to start. Although in this experi-
ment, which lasted 43 hours, the worm became gradually less
responsive to the mechanical stimulus of the brush by means of
which it was transferred to the starting point, its fatigue did not
materially affect the negative character of its response to light.
Time Required to Leave a Unit Circle. In obtaining the data
on this point, the apparatus and method already described (p. 73)
were employed. It was found that when worms of different
species were subjected to three different intensities in immediate
succession the degree of intensity did not prove to be as important
a factor as fatigue in determining the average number of seconds
necessary for the worm’s exit from a circle 10 cm. in diameter.
During the series of experiments upon this point care was exer-
cised so to vary the succession of intensities that the effect obtained
could not be attributed to any cumulative increase or decrease of
intensity. “Thus, on one day the order of intensities was I, 2, 3,
on the next 2, 3, 1, and on the third, 3, 1,2. In Table XVII the
data obtained are arranged on the left with reference to the actual
intensities employed and on the right with reference to the suc-
cession of trials made upon the various species which are desig-
nated in the middle column. The averages in the table are each
made up of four records.
It will be noted that Phagocata gracilis and Planaria gono-
cephala are, according to these figures, less subject to fatigue than
Dendroccelum lacteum or Planaria maculata.

The Reactions of Planarians to Light 83

Manner of Coming to Rest. Loeb (’93b) and others have shown
that planarians under the influence of directive light generally
come to rest in regions of lessened intensity. A few experiments
were made bearing on this point. By means of screens and back-
grounds, both black and white, a rectangular glass aquarium was
arranged so that the area of least intensity was plainly localized
and could be varied in different ways. In Fig. 8 are shown (1)
the places where worms (P. gonocephala) which had been started
together in the middle of the dish finally came to rest; (2) the num-
ber of worms in each locality; and (3) the different combinations of
backgrounds and screens used in each of the experiments.

TABLE XVII

Relative effect of fatigue (at right of table) and change in intensity of light (at left of table) as shown b
i g g §' y &£ y
the average number of seconds required for individuals of various species of planarians to leave a circle
Io cm. in diameter

Wherever shaded borders are indicated the aquarium was
surrounded on five sides by black screens and likewise on the sixth
side except for a narrow space admitting the light, the direction
of which is indicated by arrows; in a similar fashion, where
unshaded borders appear, light-reflecting screens enclosed five
sides.

It will be seen at a glance that the great majority of the worms
placed in directive light come to rest as far from the light as pos-
sible. That this is due to the directive power of light is at once
apparent by comparing 4, B and C with D, where the light was
non-directive. The darkened area was selected whenever the
directive force of the light did not prevent, as in 4, C and D,

84 Herbert Eugene Walter

The five worms coming to rest on the lighter side of D were
carefully examined and found to be mutilated or fragmented
individuals, while the same was not true of the others.

The reason why the worms in B failed to arrive in the darkened
area is probably that, being started near the middle boundary
line, their first movements were normal, 7. ¢., away from the light,
and carried them into the area of greatest intensity, whence they
were unable to escape. In this case the effect of the directive
light seems to have more than counterbalanced the locomotive

| | Light vertical

A B C D

Fig. 8 Planaria gonocephala. The arrows represent the direction of the light. The dotted areas
were surrounded by black backgrounds, except for a space on the side toward the light, and the clear
areas similarly by white backgrounds. The figures represent the number of planarians that came to
rest in any particular locality.

energy exerted by the worms. Had the species experimented
upon been Phagocata gracilis, instead of Planaria gonocephala,
the result might have been different, for in the former species, as
already shown (p. 78), the phototactic response is secondary to
the tendency to a general wandering.

It was frequently observed that worms when fatigued after a
period of activity apparently lost their phototaxis, with the result
that the final movements of a tired worm would sometimes be
made toward the light. Such behavior is probably not to be con-
sidered as a reversal of phototaxis, but rather as indifference to

The Reactions of Planarians to Light 85

photic stimuli, due to the worm’s lowered physiological state and
a chance turn toward the light. In fact the final position taken
by 49 fatigued worms with reference to the source of light, showed
that only five of them, or 10+ per cent, pointed away from the
light while 15 (30+ per cent) were headed toward the light and 29
(59+ per cent) stopped indifferently at right angles to tae tars
quite probable that among the external fictors that influence a
worm to come to a halt, light plays an exceedingly insignificant role,
as compared with the stimulus of contact or some stimulus, prob-
ably chemical, given out by other worms in close proximity.

One curious instance was observed, however, in which light
was apparently of more importance than contact or other stimuli
in determining the place of coming to rest. A large crystallizing
dish half full of water was left over night with a few planarians in

Fig. 9

it. Floating on the surface of the water in this dish was a small
Petri dish, in which a few more planarians were isolated. In the
morning the worms in both vessels were found grouped at the
same region on the outside and inside of the smaller dish, as shown
in Fig. 9.

This curious distribution on both surfaces of the Petri dish
could not be due to chemical stimulus exerted by one group of
worms on the other, and there seems to be no particular reason
why a thigmotactic reaction should have caused them to assemble
in such a way. The locality chanced to be one, however, where
the intensity of the light was considerably reduced; this seems
to offer a reasonable explanation of the observation.

Bdelloura in coming to rest shows an entirely different behavior.
When left over night free to wander in an aquarium half of which

86 Herbert Eugene Walter

had been previously covered with black cloth to exclude most of
the light, this species was found in the morning in the light area,
a behavior exactly the reverse of that shown by fresh-water pla-
narians. Another peculiarity of this species is that individuals in
coming to rest arrange themselves in compact rosettes with the
anterior end of the body pointed toward the circumference of the
rosette, while the sucker-like posterior end remains attached near
the center of the group. ‘They are so delicately responsive to me-
chanical stimuli that any slight disturbance of one member of such
a rosette is sufficient to throw the whole group into activity. The
advantage to the individual worm of such a habit of arrangement
in coming to rest, is evident.

Finally, Bdelloura was repeatedly seen on taking the resting
position to point directly toward the light with the anterior end a
the body raised and the posterior end Hatencd out into a sucker-
like expansion.

Summary. Fresh-water planarians (Dendroccelum, Planaria
and Phagocata) are negatively phototactic while Bdelloura is posi-
tively phototactic.

Negative planarians deviate most from the direction in which
they are started if pointed toward the light and least if pointed
away from the light, an intermediate deviation occurring when
they are pointed at right angles to the light.

The rate of locomotion is greater when worms are headed toward
the light than when they are headed away from it.

During successive trials the rate of locomotion decreases.

Negative planarians frequently take an apparently positive
course because the impulse to move in any direction is greater than
the phototactic impulse.

The normal negative phototaxis of a worm may change tem-
porarily to positive by reason of some physiological state ania is
not obviously referable to external stimuli.

The greater the intensity of the light the less worms wander in
their course. When they are Peaded away from the source of
light, there is less error in the precision of their orientation than
when they are started toward it.

Planarians frequently travel away from the source of light diagon-

ally instead of directly.

The Reactions of Planarians to Light 87

Bdelloura continues activity in the light for a much shorter time
than Phagocata.

When subjected to successive trials the period of a planarian’s
activity decreases.

Change in the intensity of light is less important than the effects
of fatigue in determining the time required for a worm to leave
a unit circle. When fatigued, worms often become indifferent
to light, coming to rest less frequently in an oriented position with
reference to the light than in an unoriented one.

Fresh-water planarians come to rest as far away from the source
of light as possible and, if the directive stimulus does not prevent,
in the region of least illumination.

Bdelloura candida, on the contrary, comes to rest in regions of
greater rather than of less illumination; usually worms of this
species arrange themselves in compact rosettes with the anterior
ends pointed outward.

B In Changing Directive Light

The light acting upon planarians in their natural habitat must
necessarily be a variable factor of great complexity, since its inten-
sity changes constantly throughout the day, while the position of
the sun relative to various surfaces which reflect light is also
continually shifting.

The fact that planarians, to a great extent, keep out of the light,
does not diminish the force of this statement, for whatever the
part played by light in their behavior, it must always be an exceed-
ingly varied and complex one.

Changes in the Intensity. When a worm is gliding away from
a source of light it shows a more marked response to change of '
intensity when the change is made suddenly than when it is made
gradually. In fact, it is possible by exercising patience and care to
change the intensity of directive light to a considerable degree so
gradually as to produce no corresponding response on the part of
the worm, whereas a comparatively slight change, if abruptly
effected, immediately results in the animal’s performing some one
or more of the acts in its repertory of behavior, such as halting,
wigwageging, etc.

88 Herbert Eugene Walter

In all the experiments made upon the effects of change of inten-
sity in directive light, more responses were found to occur when the
intensity was decreased than when it was increased. This is in
agreement with the experiments already described relating to the
critical region between two intensities, in which it was found that
worms show a greater number of responses when going from a
higher into a lower intensity than vice versa.

Bdelloura is particularly sensitive to changes in intensity. It
is necessary to throw a shadow on a moving worm only momen-
tarily tg cause it to perform vigorous wigwag movements or to
change the direction of its course.

Whitman (99), writing of Clepsine, suggests that the extreme
agitation of this animal when a shadow is thrown upon it may be
the result of natural selection, since any sudden shadow cast upon
it in its natural environment may be caused by a turtle swimming
overhead, to which the leech, if it is quick enough, may become
attached. It may be that Bdelloura, which is also an ecto-parasite,
has developed this extreme responsiveness to sudden decrease of
intensity in a similar way.

Changes i in Direction. The precision with which all the pla-
narians in a dish may be made to pass back and forth by shifting
a directive light from one side to another is astriking g phenomenon,
which is sure to impress anyone who sees it. By careful manip-
ulation of the light, it is possible even to make an individual
planarian follow a predetermined path in the most undeviating
manner. For example, when two lights, placed near the ends of an
aquarium, are alternately turned on and off, the worm will zigzag
across the field, at right angles to the direction of the lights, while
under a moving light it may be made to turn around and around,
almost as if its posterior end were a pivot, to trace figure 8’s and
curves of various patterns, or to turn abruptly at right angles an
imaginary corner.

Unlike the changes in intensity previously described the degree
of abruptness in any change in the direction of the light made no
apparent difference in the quale, of the reaction, since any change
in direction, however gradual,-met with an immediate response
on the part of the worm. Indeed it was necessary to abandon an

The Reactions of Planarians to Light 89

attempt to illuminate one side of the worm alone because the ani-
mal invariably turned faster than it was possible to regulate the
light.

The quickness with which this delicate response to any change
in the direction of the light occurred was found to increase upon suc-
cessive trials. A square aquarium was arranged so that it could be
illuminated instantly at either end, in a room otherwise dark.
With one light on, a planarian was allowed to move until it had
assumed the characteristic negative direction, whereupon the
source of illumination was instantly changed 180° by turning this
light off and the one at the other end of the aquarium on. ‘The
time required for the worm to become headed about was noted and
then a reversal of lights repeated and the interval necessary for re-
adjustment again recorded. Ina typical experiment of this kind
the number of seconds required by the worm, Planaria maculata,
to accomplish re-orientation were for 16 successive orientations as
follows: 260, 70, 100, 60, 65, 110, 60, 85, 70, 105, 80, 60, 50, 40,
45, 35. The sum of the first eight is 810 sec., that of the last
eight, only 485 sec.

Summary. Planarians show a greater response to sudden
change of intensity than to gradual change. ‘This response 1s
more pronounced when the intensity is lowered than when it is
rgised.

Bdelloura is particularly affected by sudden changes of in-
tensity.

Planarians respond with great precision to changes in the direc-
tion of the light, and as promptly when the change is gradual
as when it is abrupt.

The period required for re-orientation to changes in the direc-
tion of light, diminishes upon repetition.

C [In Combination with Other Responses

It is impossible to subject planarians to the influence of light
alone. ‘The best that can be done is to render extraneous factors
as uniform as possible. For example, so long as a moving worm
is kept upon a horizontal surface there can be no directive geotactic
stimulation, because the worm is moving in a plane at right angles

go Herbert Eugene Walter

to the force of gravity. The moment the worm begins to glide up
the sides of an aquarium, however, the relation of the axes of its
body to the center of the earth changes and directive geotaxis
results.

No systematic attempt was made to analyze compound stimuli,
for such a study would overstep the boundaries set for the present
inquiry. Nevertheless certain facts bearing on this point were
incidentally noted and these may properly be detailed here.

Geotaxis. Ina majority of cases, Planaria gonocephala seems,
after several hours of exposure to the dark, to be positively geo-
tactic, and after several hours of exposure to light, negatively geo-
tactic, as shown in the following series of observations.

A cylindrical aquarium jar 20 cm. in diameter and 40 cm. high
was placed before a moderately lighted window and stocked with
a freshly obtained supply of about 300 worms. No stones, sand,
or water-weeds, which would afford places of concealment, were
introduced. At intervals during the next 10 days the distribution
of the worms was recorded and these records are brought together
in Table XVIII.

TABLE XVIII

The distribution of about 300 planarians (Planaria gonocephala) in an aquarium, as observed forenoons
and afternoons during 10 days. The figures express percentages

PLACE IN THE AQUARIUM | Top SIDEs | Bottom
Time of day a.m. | p-m. a.m. p-m. | a.m. p.m.
PSRs aan en ae oncmanenoeoeede arin 5I i | | 38
PA pri o.7erotasecshsfalatesasabelel Seriorarcielee sete aeet 61 13 26
ANpral 28/25. he, grace enatanacet ere a re4ay suesetonssaveostanataiera 74 3 II Is 15 82
IMayitiese nts 72 29 6 7 22 64
IW IEW oa sanntis HPtIg OD RAB OS Criehao OO GOde. oe 63 39 12 | 20 25 41
IMA ypegictstere cia any aitianaxagass ts ahsuive sigiersiaseia\elaters 67 43 16 | 41
May 4.. 50 16 | 34
DMA yi Sara stoseyeteve/=tsferere' foie st eeratsista etotosehs 6 eles e 31 13 56
——— |
ISVET APES yaye ye Soe seiko michele Sie si siete 60.5 30.6 12-3). |j| UI¥-20 || e27 ee 56.2

‘The forenoon census was taken about 8 o’clock, when the worms
were re-arranging themselves after the darknessof the night, while
the afternoon records were made about 4 o'clock, when the worms

The Reactions of Planarians to Light gi

had been all day in the light. The average at the bottom of the
table indicates, first, that an approximately equal percentage of
worms was found on the sides of the aquarium at both times of day,
which may therefore be left out of the reckoning, and, secondly,
the occurrence of a significant migration during the interval be-
tween 8 a. m. and 4 p. m., demonstrated bythe distribution of the
worms at the top and bottom of the jar respectively. Accord-
ing to the data obtained, at least 30 per cent of the worms in the
top group must have become positively geotactic and gone to the
bottom during the day.

A later set of experiments in which an aquarium was kept
swathed in black cloth during the day showed less migration.
The conclusion naturally follows that geotaxis is more likely to
occur in the presence of light than in its absence. Whether there
is a regular diurnal vertical migration among planarians in nature,
as Birge (’97) and Schouteden (’02) found to be true for fresh-
water entomostraca, and various authors‘ for different forms in
marine plankton, remains unknown. It is probable, however,
that planarians ordinarily remain quiescent on the under sides of
stones or in other shaded places for considerable intervals of time,
coming under the influence of light only when started into activity
through some other stimulus.

A worm placed in an aquarium with square sides and left free
to travel undisturbed on the bottom or the sides occupies the
sides more frequently than the bottom.

In a trial to test this point, an aquarium was used, the bottom
area of which measured approximately five times that of the sides.
The course pursued in this aquarium by one worm (P. gonoceph-
ala) in directive light and covering 1340 cm., was plotted and the
percentage of distance traveled on the sides was found to be prac-
tically equal to that traveled on the bottom, notwithstanding the
fact that the animal was started in the middle of the bottom, where
it had five times as much available territory to travel over as on
the sides. Other things being equal, therefore, this worm showed
itself five times as ready to travel on the sides of the aquarium as on
the bottom.

*Groom and Loeb (’g0), Loeb (’93a), and Parker (‘o2).

g2 Herbert Eugene Walter

The existence of such a decided geotactic tendency should not
be forgotten when trying to determine the part light plays in
planarian behavior.

Again, it was found that there was less accuracy of orientation
to directive light while the planarians were on the sides of the
aquarium in a position parallel to the light rays than while they
were on the bottom.

Their behavior in the former case was the resultant of at least
two known stimuli, gravity and light, whereas gravity was prac-
tically eliminated when they elided on the floor of the aquarium.
In the experiment cited under the preceding paragraph 92 per
cent of the distance traversed by the worm on the bottom of the
aquarium was in a direction in general away from the light, as
contrasted with only 79 per cent when it was traveling on the sides
of the aquarium. This difference of 13 per cent may represent
roughly the necessary correction for geotaxis, in order to ascertain
the influence of light alone.

Thigmotaxis. Contact with the substratum is an almost con-
stant condition of planarian activity. Occasionally worms may
be seen dangling free at the end of a mucus-thread, as commonly
occurs among many fresh-water snails; sometimes they may fall
helplessly from the surface-film to the bottom, but definite con-
tact with something firm is the rule during their ordinary loco-
motion.

A change in the degree of this contact, and consequently a pro-
duction of thigmotactic stimulation, may come about in two ways
the surface on which the animal glides may present irregularities,
such as increased roughness or a different degree of solidity, or
the worm itself may vary in the extent of Cae -surface which it
brings into contact with the substratum. ‘This latter method of
causing thigmotactic stimulation applies especially to Bdelloura,
which has the habit of frequently alternating a leech-like looping
movement with ordinary gliding, thus ae its contact rela-
tions and probably producing a Antec ppling. in conse-
quence.

As already mentioned, Bdelloura, when subjected to sudden
dark, usually detaches itself from its support and wriggles vio-

The Reactions of Planarians to Light 93

lently in the water. It is uncertain how far this behavior is attrib-
utable to light alone or to some combination of light and thigmo-
taxis.

This phenomenon of compound stimulation occurs in a less pro-
nounced way whenever a change of light intensity results in the
“wigwagging” response common to planarians. ‘The same uncer-
tainty prevails as to how far the subsequent behavior of the worm
may be due to the direct stimulation of light and how far to thig-
motactic stimulation primarily and to hal stimulation second-
arily. It is evident, then, that under any circumstances there is
such a close interrelation of stimuli that an accurate analysis of
the consequent behavior 1s difficult.

Further evidence of the close relation between different kinds
of stimuli is afforded by the fact that planarians are more respon-
sive to the mechanical stimulus of a slight jar when the entire ven-
tral surface of the body is in contact with the substratum than
when the anterior end is lifted up and waving about. Apparently
the greater the degree of contact the greater is the effect of a jar-
ring mechanical stimulus.

This point was demonstrated by means of a small aquarium
mounted on a turntable, such as is used in “ringing”? micro-
scopic slides, in such a way that it could be rotated with great
ease and delicacy. A light from one direction only was projected
upon the single pianeian placed in the aquarium. Any attempt
to change the. angle of light by rotating the aquarium ever so slightly
pesnltecl instantaneously i in a momentary halt on the part of the
worm, provided it happened to be gliding with its ventral surface
entirely in contact with the floor of the dish. If, however, the
rotation was made when the anterior end of the worm was lifted,
the halt did not so readily occur. This response was of such deli-
cacy that with a little practice it was possible to halt the anterior
end of a worm without disturbing the continuous progress of the
posterior end! That this halting was due to thigmotaxis rather
than to any rheotaxis induced by the movement of the animal
against the relatively stationary water particles, is shown by the
fact that the reaction was more pronounced when the anterior
end of the body was held flat than when it was raised and so brought
more under the possible influence of a water current.

94 Herbert Eugene Walter

Finally, it may be recalled that in a preceding section data were
given (Table X, p. 68) to show that there is more response to light
while worms are upside down on the surface-film than when they
are in contact with the bottom of the aquarium, a difference
probably referable in large measure to the different thigmotactic
relations in the two cases.

Gomiotaxis. Goniotaxis is a term introduced by Pearl (’03, p.
561) to define a particular kind of thigmotactic response in which
the “different parts of the body are brought into such positions
that they form unusual angles with each other,” as when a plana-
rian occupies the angle formed between a side and the bottom of
an aquarium.

There is no doubt that the peculiar movements resulting from
the goniotactic stimulus directly modify the phototaxis of the
worm. Once in the angle of an aquarium a planarian becomes
increasingly indifferent to light. In one series of records, show-
ing how a considerable number of planarians came to rest, it was
found that the majority came to rest in an “‘angle” and that out
of this number 78 per cent failed to orient to the light. The stim-
ulus of the “angle” was greater apparently than the stimulus of
light.

Furthermore, it is to be noticed that goniotaxis is always more
effective if the worm is in a lowered rather than ina heightened
physiological state, for whenever a planarian is freshly intro-
duced into an aquarium and is in an aroused condition on
account of the mechanical stimulation necessarily given it in
transference, it will pass over angles and crevices with total
indifference, all the while responding plainly to light. As soon
as it has become fatigued, however, if its path chances to cross
an angle or crevice it exhibits goniotaxis at once by slowing down
and remaining in the new situation, as if caught in a trap, with
complete disregard of the continued action of directive light.

Chemotaxis. Pearl has made an extensive study of this phase
of planarian behavior and suggests that the well-known planarian
habit of collecting in groups may be explained on the supposition
that a resting planarian is surrounded by a halo of chemical ema-

The Reactions of Planartans to Light

\O
al

nations which serve as a direct stimulus to other planarians, attract-
ing them and causing them to come to rest in groups.

In this connection it is worth mentioning that several times
when Dendroccelum lacteum was put im an aquarium with other
species of planarians, the individuals of this species would later be
found gathered into aseparate group bythemselves. “This manner
of isolation was also repeatedly noticed in examining on the under
sides of stones taken from the pond at Falmouth, Mass. A similar
segregation of species in the case of P. alpina and P. gonocephala,
was noted by Collin (’91). He says (’g1, p. 180) “‘liyjima fand
diese beiden Arten zusammenlebend, wahrend sie im Harz stets
getrennt vorkamen; auch in der Gefangenschaft schien die P.
alpina die grossere P. gonocephala in demselben Behalter zu
meiden und thr angstlich auszuweichen.”” It would be difficult to
explain how these planarians avoid each other so as to fraternize in
this fashion, except on the basis of some delicate chemotactic
response which caused them to halt when they entered the chem-
ical halo of their own kind, but not to do so in the different chem-
ical halos of other species. As in the case of goniotaxis, the mani-
festations of phototaxis may be entirely superseded by the effect
of feeding (Chemotaxis). When once a hungry planarian, driven
by directive light into the neighborhood of a crushed snail, becomes
subjected to the chemical stimulus arising from the fluids of that
object as they are disseminated through the water, it seems to
become suddenly indifferent to the light, owing to the greater
influence of the chemical stimuli.

The same inhibition of the influence of light by a chemotropic
response to food has been observed by Parker ('03) on the mourn-
ing-cloak butterfly, Vanessa antiopa L. He says (’03, p. 457)

“when a butterfly alights on a bough, it orients in the sunlight
with the usual precision. Should the sap be running from a near
stem, the insect is very soon attracted to the spot, begins feeding,
and moves about from that time on with no reference to the direc-
tion of the sun’s rays. “Thus, when feeding or near food the but-
terflies do not respond phototropically.”’ Furthermore Darwin
(81, p. 23) observed that earthworms are less disturbed by light
while feeding or during copulation than at other times.

go Herbert Eugene Walter

The foregoing examples illustrate only a few of the many modi-
fications of light responses due to the interference of some other
stimulus.

Summary. In judging the effect of any stimulus upon an ani-
mal it is necessary to have constantly in mind the accelerating or
inhibiting effects of other stimuli which may be influencing the
organism at the same time. In the case of planarians some ai the
responses known to be intimately connected with phototaxis are
geotaxis, thigmotaxis, goniotaxis and chemotaxis.

Planaria gonocephala shows itself to a certain extent negatively
geotactic after several hours of dark and positively geotactic after
a similar interval of light.

When given horizontal and vertical surfaces of equal extent,
worms travel more on the vertical surfaces.

Their accuracy in orienting themselves to light while subjected
to geotactic stimulus on a vertical surface is less than when they
are traveling on a horizontal surface, where the directive geotactic
stimulus is eliminated.

Thigmotactic stimulus may result either from an environmental
change in the substratum, or a change in contact caused by the
worm itself whereby its relation with the substratum is varied.

There is a close interdependence of the various stimuli which
may be acting on an animal at the same time.

Behavior may be the direct consequence of light or the indirect
result of light combined with the direct effect of a thigmotactic
stimulus indirectly brought about by some change in the intensity
of the light.

The greater the degree of contact with the substratum the more
responsive a planarian becomes to the mechanical stimulus of jar-
ring, but the less to the stimulus of light, as shown by comparing
the behavior of worms on the surface film with their behavior on
the aquarium floor.

Goniotaxis has an inhibitive effect on phototaxis; this effect
becomes more apparent as the worm reaches a condition of fatigue,
phototaxis meanwhile becoming less apparent.

Dendroccelum lacteum exhibits a remarkably delicate response
(Chemotaxis ?) in frequently coming to rest in the neighborhood
of its own kind.

The Reactions of Planarians to Light 97

Hungry planarians in the presence of food have their photo-
taxis entirely obscured.

3 KINDS OF BEHAVIOR

In the two preceding sections, treating of Photokinesis and Pho-
totaxis, respectively, animal behavior, as illustrated by the effect
of light upon planarians, has been taken up from the point of
view of the stimulus. In the two following sections, on the other
hand, the reactions of planarians will be dealt with from the stand-
point of the animal rather than from that of the stimulus.

To this end a classification of the behavior of planarians in light
is here presented based upon (A) generic and specific differences,
and (B) individual differences.

That there are morphological differences which fall naturally
within the lines of this classification has long been recognized,
indeed, the criteria used in classification by systematists are based
almost exclusively upon such differences, while relatively little
importance has been attached to differences in the behavior of
animals.

As already mentioned in the historical review, Loeb (94), in
dealing with the differences of behavior which characterize the
two genera, Planaria and Thysanozoon, pointed out that decided
physiological variation may appear in forms closely related mor-
phologically. The same fact had been previously emphasized
for the case of the pulmonates by Willem (’g1). Obviously such
physiological variations do not furnish reliable criteria for the
systematist, since they are so largely dependent upon environ-
mental causes, and furthermore the work of the systematist is
usually done upon dead animals. Nevertheless some interesting
relations between behavior and systematic position await the stu-
dent who approaches the study of animal behavior from this direc-
tion.

Strictly speaking, all behavior is individual behavior. In this
sense it is manifestly incorrect to speak of the behavior of a genus
or of a species per se.

The behavior of individuals may, nevertheless, be classified into
responses which are characteristic of all the members of a genus,

98 Herbert Eugene Walter

or again into responses which are characteristic of only one species
of a genus and not necessarily of other species of the same genus,
and, finally, into those peculiar to the individual as such, which
may not in all particulars be shared by other representatives of
the species to which the individual in question belongs. It is in
this sense of the terms generic, specific and individual, that behav-
ior will be taken up in the present section.

A Generic and Specific Behavior

In the present inquiry a basis for generic comparisons is afforded
by a study of the behavior of edad: of four different genera,
namely, Planaria, Dendroccelum, Phagocata and Bdelloura, while
some idea of specific differences is ee possible by compar-
ing the behavior of individuals of the two species Planaria macu-
Tet and Planaria gonocephala. In the cases of Dendroccelum,
Phagocata and Bdelloura it is obvious that the conclusions drawn
are based in each instance upon the behavior of representatives of
a single species under each genus. ‘The question may be properly
raised as to how far such conclusions indicate generic behavior
and how far specific behavior. Conceding that from the data
obtained exact deductions may not be drawn, the fact still remains
that the three species, Dendroccelum lacteum, Phagocata gracilis,
and Bdelloura candida, are separated from each other by generic
gaps, such that the differences exhibited by these species may be
regarded as generic in degree. The point unestablished, then, is
whether other species of the genera in question if examined might
not show that the behavior, which in these single representative
species seems generic in nature, is not characteristic of other species
of the same genus as well.

It will be convenient to present the data of both generic and
specific behavior at the same time.

Percentage of Negativeness. The manner of obtaining this
_ criterion of behavior has been explained in the section on
Phototaxis (p. 72). It will be remembered, too, that in Table
XII a comparison was made between positive and negative worms,
showing the degree of their orientation to directive light. The

The Reactions of Planarians to Light 99

data there used are rearranged for the present purpose in Table

XIX.

TABLE XIX

Percentage of generic and specific negativeness in worms started at right angles to incident light, as deter-
mined at the circumference of a circle 10 cm. in diam. by the average amount of their deviation from

the directions in which they were started

SPECIFIC

GENERIC DIFFERENCES
DIFFERENCES

F Planaria
Mende eee Boe hpi aried |iEdellowealy seca gono-
coelum cata maculata
| cephala
Number of observations........... 78 80 158 10 78 | 80
Total number of degrees positive... 155 238 165 397 5 | 160
Total number of degrees negative...) 2112 1964 4070 50 | 2102 | 1968
Percentage of negativeness........ 93-1 89.6 96.1 II 99-9 84.6

Comparing the figures given in this table, a greater range of
difference is seen to obtain between the two species of Planaria
(P. maculata and P. gonocephala) than between the genus Planaria
and either of the other negative genera, namely, Dendroccelum and
Phagocata. Although not indicated in this table, similar results
appear when the number of times the worms went in a negative
direction is used as a basis of comparison, instead of the total num-
ber of degrees of negative deviation.

Character of the Course in Directive Light. When worms were
placed on the middle of a rectangular aquarium floor and sub-
jected to a directive light their movements showed both generic
and specific differences. By experimenting with one worm at a
time it was possible to plot on a sheet of paper with sufficient
accuracy for general comparison the entire course of the worm
during a considerable period. This was done many times and
typical records of such observations are given in Figs. 10-14. — In
such instances the worm was exposed to a light of approximately
147 c.m., placed so as to correspond to the right side of the
figures. The central rectangular area bounded by the broken lines
indicates the limits of the floor of the aquarium, while the smaller
exterior adjacent areas represent its vertical sides so rotated as to

100 Herbert Eugene Walter

bring them into the plane of the floor. The course taken by a
planarian is indicated by the tortuous line. The full line shows
the course taken on the solid surface of the aquarium; the dot-

Fig. 10 Dendroccelum lacteum. The shortness of the path shows comparatively little persistence

in locomotion, and the direction, considerable indifference to the source of light.

and-dash lines, the course of the worm on the surface film. The
dotted line indicates a hiatus in the path, made necessary by the
attempt to represent on a flat surface a continuous line which tra-
verses vertical as well as horizontal surfaces. A succession of

The Reactions of Planarians to Light IOI

abrupt kinks in the line signifies that at that point the worm exe-
cuted decided wigwag movements with its anterior end. The
figures are reduced in size from the original records.

In Fig. 10 is given a specimen record of a Dendroccelum’ which

Fig. 11 Phagocata gracilis. This path shows great activity on the part of the worm, and, although
it is mostly laid down away from the source of the light, it shows that the worm experienced no great
difficulty in moving toward the light.

came to a standstill after 18 minutes of locomotion. ‘The first
movement of this worm was diagonally away from the light, but
It soon came back toward the light traversing almost the entire

102 Herbert Eugene Walter

width of the aquarium and in doing so showed considerable indif-
ference to the directive influence of the light. Its susceptibility
to goniotactic stimulus is plainly shown by its behavior upon reach-

Fig. 12 Bdelloura candida. This path was traversed with much ‘‘wigwagging;” there was indif-
ference to the source of light and locomotion was not of long duration.

ing the angle formed at the junction of the sides and floor of the
aquarium, as well as by its manner of finally coming to rest.
A typical Phagocata (Fig. 11), on the other hand, exhibited

The Reactions of Planartans to Light . 103

almost no goniotaxis, although the worm repeatedly crossed
the line of the angle. The response to the directive influence
of the light, too, was in this case even less than that of the

Fig. 13 Planaria maculata. Considerable activity was shownover this course and a decided

inability to approach the source of the light beyond about the middle of the aquarium.

Dendroccelum just described, as is evident from the general wan-
dering character of the course. Although the Phagocata in ques-
tion frequented both sides of the aquarium—that which was toward

104 Herbert Eugene Walter

the light, as well as the opposite side—its wanderings were in the
main on the side away from the light. An hour’s activity is chron-
icled in the record, at the close of which the worm was apparently
as energetic as ever.

Fig. 14 Planaria gonocephala. The path is of the same generic type as with Planaria maculata
(Fig. 13), and is easily distinguishable from those of Dendrocelum, Phagocata and Bdelloura.

Fig. 12 gives a characteristic record of the way in which Bdel-
loura behaves. The first movement of this specimen was more

The Reactions of Planarians to Light 105

toward than away from the source of the light, but very soon wig-
wagging motions set in, and after every exercise of these move-
ments, which were apparently in the nature of explorations, a
change in the direction of the course was effected. As might be
expected, such abrupt changes in direction were more difficult of
execution when the worm was on the surface-film.

Characteristic movements of individuals of the genus Planaria
are shown in Figs. 13 and 14. From these two typical records it
would be difficult to select any diagnostic points which would dis-
tinguish the behavior of P. gonocephala from that of P. maculata.
There is no doubt, however, that taken together the behavior of
representatives of these two species presents a distinct (generic) dif-
ference from that of the representatives of the other genera studied.
The most striking feature of the Planaria records ( (Figs. 13 and 14)
is the high degree of response exhibited by members of this genus
to the decane action of light. Although many attempts were
made by the individual worms to penetrate the half of the aqua-
rium nearer the light, yet they seemed as unable to keep to that
direction as they would have been had a solid barrier been inter-
posed between them and the light. This characteristic respon-
siveness to directive light helps to explain why (as shown in Fig. 8,
B, p. 84). P. gonocephala was unable to come! to rest In the area of
lessened illumination as it would naturally have been expected to do.

From the cases cited in this section, at least, it may be afirmed
that the generic differences are so pronounced that one could
take a miscellaneous, unidentified assortment of such records
and correctly assign the great majority of them to the proper
genera.

Duration of Movement. When worms of different genera are
subjected to the same light intensity there is considerable variation
in the time required to bring them to a standstill. Bdelloura is
usually the first to stop, followed in order by Dendroccelum, Pla-
naria and Phagocata. Of the two species of Planaria, P. gono-
cephala, although averaging somewhat smaller in size, usually
keeps in motion for a longer time than P. maculata. The individ-
ual records of the duration of movement given in Figs. 10-14
may be taken as a typical set of records. “They were as follows:

106 Herbert Eugene Walter

Fig. 10. Dendroccelum, 18 min.

Fig. 11. Phagocata, 60 min. (still moving).

Fig. 12. Bdellourz, 15 min.

Fig. 13. Planaria maculata, 47 min.

Fig. 14. Planaria gonocephala, 60 min. (still moving).

Woodworth (’97) in contrasting the activity of Planaria macu-
lata, P. gonocephala and P. dorotocephala states that individuals
of the latter species remain in motion longer than individuals of
the other two—an observation confirmed by Pearl (’03).

Degree of Wandering. fa worms started at the center of a
circle parallel to the direction of the light and pointing away from
its source, then the more devious its course the more it may be said to
wander. Both generic and specific differences were obtained bear-
ing upon this phase of behavior. Selected instances of such dif-
ferences are given in Table XX, expressed in average degrees of
deviation upon emergence from a circle 10 cm. in diameter.

TABLE XxX

The average generic and specific differences between individuals of four genera and two species of
Janarians expressed in degrees of deviation upon leaving a circle 10 cm. in diam. In ever
y

instance the worm was started away from the source of the light

SPECIFIC
GENERIC DIFFERENCES
DIFFERENCES
Ded Pl PI _ | Planaria
os 248°" | Planaria Bdelloura ae gono-
celum cata maculata
cephala
Average degree of deviation....... 9-4 30.0 24.6 132.0 21.6 27-7
Number of observations........... 56 46 gz 10 46 46

The remarkably large deviation shown by Bdelloura is due to the
fact that it is a positive worm. When pointed toward the light
its deviation was only 39.3°, a number which would perhaps be
more justly comparable with the other records in this table. But
even so, it will be seen that Bdelloura, of all the forms observed,
is the least oriented by directive light. Specific differences in
the degree of wandering are in general less marked than the
generic differences, according to the records in Vable XX.

The Reactions of Planarians to Light 107

Rate of Locomotion. As regards rate of locomotion the records
of specific differences exhibit a wide range, although not as great
as that of the generic differences existing between Dendroccelum
and Phagocata.

TABLE XXI

The average rate of locomotion expressed in millimeters per second

SPECIFIC

GENERIC DIFFERENCES
DIFFERENCES

Dendro- Planaria | Planaria

Phagocata | Planaria

celum maculata |gonocephala

Rateinvmm. per seG...feincslsteieesis|-)2)- 1] (O50 1.395 1.272 1.470
Number of observations........-..... 40 40 80 40 40

It is interesting to note in this connection that Parker and Bur-
nett (’00, p. 385) give the average rate for Planaria gonocephala
as 1.08 mm. per sec., while Pearl (’03, p. 546) records for P. macu-
Jataler: 48 mm. per sec. in the case of a worm Ir mm. in length
and 1.23 mm. per sec. for one 6 mm. in length. An average
of these two records, that is, 1.355 mm. per sec., might per-
haps be comparable with the average (1.272) given in T able XXI,
since an equal number of large eed small worms from each
genus formed the basis on which these averages were calcu-
lated.

Time Required to Leave a Unit Circle. If planarians invari-
ably took a straight radial path in going from the center to the
circumference of a circle, the time required to leave a unit circle
might be used in computing the rate of locomotion. Such a path,
however, is not taken. Nevertheless, records of this kind, although
untrustworthy for purposes of accurate calculation, furnish a relt-
able criterion for the comparison of generic and specific behavior.
The differences in behavior in the representatives of three genera
and two species are given in Table XXII. Bdelloura failed so
frequently to emerge from the circle that it is excluded from the
list. Each genus and species was tried an equal number of times
in light of three different intensities.

108 Herbert Eugene Walter

As might be expected, Table XXII presents a close parallelto
Table XXI. The only difference in the relative values of behav-
ior, expressed by the averages of rate and time in these two tables,
appears in the case of P. maculata. ‘This species, though first in
the scale as regards actual rate of locomotion, is second as regards
the time required to leave a unit circle, a condition indicating
relatively more wandering on its part than was shown by any of
the other worms.

TABLE XXII

The average time in seconds taken in passing from the center to the circumference of a circle 10

cm in, diameter in directive light

Average number of seconds. . . 62 | 40 54 | 47 60

Number of observations...... 120 120 240 120 120

The Effects of Fatigue. ‘Yo obtain an idea of generic and spe-
cific differences in the effects of fatigue, two sets of averages have
been combined. First the average rate of ten worms of each kind,
when subjected to four successive trials, was first ascertained and
the difference between the first and the fourth rate was then ex-
pressed as a percentage of increase or decrease in rate, as the
case might be. Secondly, the time required to leave a unit circle
in twelve successive trials was next recorded and the average
percentage of increase or decrease in time of the last four trials,
as compared with tne first four trials, was computed. By com-
bining these two kinds of percentages the relative differences
in the effects of fatigue upon the individuals of the various genera
and species, are clearly brought out.

If the results of this computation be compared with the con-
clusions reached in another way under the preceding paragraphs
on ‘‘duration of movement,” it will be seen that there is a com-
plete agreement in the relative behavior of the different genera
and species. ‘That is, the worms most subject to fatigue are the

The Reactions of Planartans to Light 10g

first to come to rest and those least affected by fatigue continue
longest in motion
TABLE XXIII

The average generic and specific difference in fatigue

GENERIC DIFFERENCES SPECIFIC DIFFERENCES
Dendro- ’ Planaria Planaria
Phagocata Planaria
ceelum maculata gonocephala

Percentage of change in rate of

the fourth trial as compared

Withrthesfirstasj/sjcrcisieies ie) — on +9.5 i) =13 i
Percentage of change in time

required to leave a unit circle

of the last four as compared

with the first four of twelve

consecutive trials.......... 2) —10 —15.8 =23 —8.7
Average percentage of fatigue =22 —0.25 —12.4 —18 = 6.5
Total number of comparisons 30 20 40 20 20

Responses to Changes in Intensity. When worms in non-direc-
tive light pass from a given intensity to one 2} times as great,
decided differences appear in their behavior, the generic differences
being plainly of wider range than the specific.

TABLE XXIV

Average differences in response at the critical line separating two areas of non-directive light of which
one (82.50 c.m.) is approximately 2% times as great as the other (33.16 c.m.)

GENERIC DIFFERENCES SPECIFIC DIFFERENCES
|
Dendro- : | Planaria | Planaria
Phagocata _ Planaria }
celum | maculata |gonocephala
| |
Number of observations. ..... 45 | 202 | 206 50 156
Percentage of responses....... 17 37 52.5 55 50
Percentage of failures to re-
SPO Glee respec tartar steyacaidiaicye 8 63 47-5 45 50

In this table Dendroccelum is shown to respond in only 17 per
cent of its passages across the critical line separating the two dif-
ferent intensities of light, while Phagocata responds in 37 per cent

IIo Herbert Eugene Walter

and Planaria in 52.5 per cent of the cases. ‘These are differences
in degree of response that are great enough to be of unquestion-
able significance. When, however, Planaria maculata and Pla-
naria gonocephala are compared in the same way, only a slight
difference in the degree of response, namely, that between 55 per
cent and 50 per cent, is to be observed.

Manner of Coming to Rest. Although little attention was paid
to this point during the series of observations taken up for the
present study of planarian behavior, still a few indications of gen-
eric difference in the manner of coming to rest appear from the
foregoing data. Bdelloura, it will be remembered, has a distinc-
tive manner of coming to rest in close rosettes within an area of
increased illumination, while Dendroccelum shows a considerable
tendency to collect in exclusive companies during periods of inac-
tivity. With regard to the two species of Planaria studied, nothing
at all definite was observed in this connection which could be
called a true specific difference in behavior. Between Dendro-
coelum and P. gonocephala, however, a decided generic difference
seems to exist, as several series of records on orientation in direc-
tive light show. Dendroccelum according to these records came
torest in an unoriented position in 70 per cent of the cases observed,
while P. gonocephala failed to take up an oriented resting position
in only 59 per cent of the observations. In other words, P. gono-
cephala is more liable to come to rest in a position oriented with
reference to the light than Dendroccelum.

Summary. The essential points brought out in the foregoing
section are condensed for the sake of brevity and clearness in
Table XXV.

In certain instances, namely, in changes in the character of the
course (2), the influence of fatigue (7), and the percentage of
responses to change in light intensity (8), specific behavior shows
a more intimate correlation than generic behavior, otherwise the
range between the behavior of P. gonocephala and P. maculata is
greater than the generic differences separating Planaria from the
other genera under observation.

It might be expected a prior: that generic differences would
exhibit a greater range than specific differences and that similarly,

The Reactions of Planarians to Light III

specific behavior would include more phases of action than indi-
vidual behavior.

In the present series of records hardly enough representatives
of different genera and different species were under consideration
to establish any convincing generalization on this point.

TABLE XXV

Comparisons in behavior, generic and specific

SPECIFIC
GENERIC DIFFERENCES
DIFFERENCES
CRITERION OF BEHAVIOR ? Planaria
Dendro- ; Planaria
Phagocata| Planaria Bdelloura | gono-
celum maculata
| cephala
(1) Percentage of negativeness 93.1 89.6 92.2 II 99-9 | 84.6
(2) Character of course in di-
rective light
without — with great with with
Turns toward the light much indiffer- much ease No | contrast
difficulty ence difficulty |
Shows goniotaxis...... plainly slightly slightly none slightly slightly
Wigwag movements ... few few few many few few
(3) Average duration of move-
MENGE jee cise acl oe cee. 18min 60+min. ? 15 min. 47min. 60+min.
(4) Amount of wandering... . 9-4 30. 24.6 39-3 21.6 27.7
(Av. deviation in degrees) !
(5) Rate of locomotion ....... 0.85 1.395 1.270 ? 1.470 | 1.075
(In mm. per sec.) |
(6) Seconds required to leave |
axzocm. circle.......... 62a 40 54- ? 47 60
(7) Comparative influence of
fatigue, percent....... 22 0.25 12.4 ; 18 6.8
(8) Percentage of response to
change in light intensity.) 17 37 §2.5 2 55 50
(9) Manner of coming to rest.) in dark in dark in dark in light in dark in dark
in exclu- in rosettes |

sive groups |

B Individual Behavior

The analysis of specific behavior leads to the study of individ-
uals, since it is the average activity of different individuals that
makes up the behavior typical of the species. In biological litera-

T12) Herbert Eugene Walter

ture animal behavior, particularly among the lower forms, is
ordinarily referred to in its specific or even generic aspect. “The
distinctive actions of individuals, as such, it seems, have usually
been outside of the purpose of the observer.°

Individuals, however, even among such comparatively simple
forms as planarians, do not always act with machine-like uniform-
ity. Until it is possible to predict with exactness what behavior will
result under any given set of conditions, an accurate knowledge of
the behavior of any kind of organism must be based upon repeated
observations of individuals as such rather than as representatives
of species and genera.

Individual variations in behavior constantly appeared through-
out the course of the present investigation. It will be sufficient,
however, for the purpose of making clear their importance to cite
only a few instances of such variations.

It should be noticed that whenever “exceptions to the rule” of
behavior occur, as in the case of negative planarians coming to
rest in the light or Dau Poritvely phototactic for a time (see
the two cases cited on p. 78), they are ordinarily simply abnor-
mal cases of individual elavior standing out against the back-
ground of average specific or generic behavior. Exceptional cases
of this kind, however, are not so typical of what really constitutes
individual behavior as the less aberrant actions making up the
majority of the movements which the animal performs.

The main point to be recognized, then, is that the individual
presents unknown factors, which, even in the simplest forms of
life, where the range of variation is least, have never yet been
reduced entirely to hence: physical terms, a fact which i impairs
somewhat the conclusions of those writers who would draw a
complete parallel between an organism and a machine.

Rate on Successive Days. When the rate of locomotion of cer-
tain isolated individuals is averaged from four trials, for example,
and the same experiment is repeated on the following day with the
same individuals, thereby eliminating the effects of fatigue, under
as nearly identical conditions as possible, the two sets of figures

°Frandsen (’o1), who was impressed by individual differences in the phototaxis of Limax, and

Smith (’02), who worked with the earthworm, are exceptions to this generalization.

The Reactions of Planarians to Light 113

thus obtained show more variation than would be expected if the
organisms experimented upon responded in a machine-like way.
If not all, at least a part of this variation may, then, be due to
differences in individual behavior.

TABLE XXVI

The differences among tsolated individuals of different species in the average rate of locomotion, based

on 4 trials each on each of two different days, expressed in mm. per sec.

Dendrocelum Phagocata Planaria Planaria
lacteum gracilis maculata gonocephala
INTs Ohi: gaan do oodanouocenonoun 1.52 1.22 1.76 0.67
niscentlie rl Aoheacanrn qonAaads0 dpe 0.70 0.96 ay 8 0.73

The Relative Value of Individual Behavior. In the three fol-
lowing tables individual behavior will be compared with light
intensity with respect to (1) rate of locomotion, (2) range of rate
and (3) manner of turning.

First, the individual behavior of 10 worms belonging to the
species Planaria gonocephala under all intensities of non-direc-
tive light showed greater range in the rate of locomotion than the
average behavior of the same ten worms showed under any single
intensity of non-directive light.

TABLE XXVII

The relative effect on rate of locomotion of individual behavior and light intensity. The averages

are expressed in mm. per Sec.

A Variation or INpIvipUAL BEHAVIOR

Identification number of worm ..............-- I 2 3 4 5 6 7\ 8 9 10

Average rate in all the intensities given in B..... 0.79|0.57.0.70 0.64 0.83 0.700.72 0.60'0.49 0.62
e

Range = 0.34 [0.83 (No. 5) —0.49 (No. 9) ]

B Variation IN Dirrerent LicHr INTENSITIES

Light in candle meters ......... °o | 0.94 ial 39 78 126 155 | 217 | 43%

Average rate of the 10 worms given
misao deine oo coc adonepsonaeey 0.57 | 0.66 | 0.69 | 0.75 | 0.64 | 0.65 | 0.69 | 0.70 0.63

Range = 0.18 [0.75 ‘39 cm.) — 0.570 (0 c.m.) |

IIl4 Herbert Eugene Walter

Secondly, the range between the maximum and minimum rates
of ten individuals in all intensities of non-directive light was
greater than the average range of rate of the same individuals
under different intensities of non-directive light.

TABLE XXVIII

The relative effect of individual behavior and light intensity on the range of rate of locomotion,

expressed in mm. per sec.

A VariaATIoN or INDIvIDUAL BEHAVIOR

Identification number of worm............... I 2 3 4 5 (SOS; 8 9 | 10
Maximum rate in all intensities given in B..... 2.58 1.67/2.00 1.67/2.17 2.08 2.20 1.78 1.82 1.58
Minimum rate in all intensities given in B..... 0.92 0.67/0.92 0.83/1.03 0.42/0.67 0.28,0.28 0.75

AN Pe? Obst Aterstaysrarsiseare slsistaratersrotle,o}sjexstors 1.66 1.00)1.08 0.841.14 1.66 1.53 1.50 1.54 0.83

Range 0.83 = [1.66 (No. 1 or 6) — 0.83 «No. 10)]

B_ Variation IN DirrereNT Licut INTENSITIES

Light in candle meters ......... ° 0.94 | II 39 78 126). |) USS. A217) (0 43T

Maximum rate for all worms given

ee cB ee HBOS ee OMe 1.§8 | 2493} 1292 | pTL77 |) W92s| -79)) 1.87.) 200%) res
Minimum rate for all worms given

Trna A terer fats letelaie arate eidavertsewsstacints 0.71 | 1.07 | 0:74 | 1.04 | 0.77 | 0.62 | 1.15 | 1.03 | 0-79

Rangsiafirate’sjesaic.+:s;sto,<f=%6 0.87 | 0.86 | 1.18 | 0.73 | 1.15 | 1.17 | 0.72 | 0.97 | 0.76

Range 0.46 = [1.18 (11 c.m.) — 0.72 (155 c.m.)]

Thirdly, with respect to clockwise or contra-clockwise turnings,
individual factors were found to be of more importance than
differences of intensity of non-directive light in determining the
direction of turning.

It should be added that the ten worms concerned in the three
preceding tables were as similar in size and external appearance
as it was possible to select.

A Cave Planarian. This specimen came from an Indiana
cave, where it probably had always lived in darkness up to the
time of its capture. When first made the subject of experiment,
it could be briefly described as a white worm, about 6 mm. in

The Reactions of Planarians to Light 115

length, devoid of any dark pigment except in the two eye spots.
Although most nearly resembling Dendroceelum lacteum_ in
color, it showed some differences from this species in the contour
of its body and particularly in its behavior. It was thought prob-
able, therefore, that this was a representative of some species
peculiar to a dark habitat. The absence of sexual organs made
its exact identification impossible. In the present connection it
will be referred to simply as “the cave worm.” As a unique sub-
ject for the study of individual behavior, it proved to be very

TABLE XXIX

The relative effect of individual behavior and light intensity on the direction of turning, expressed

in a ratio of contra-clockwise to clockwise movements

A Variation or InpDIvIDUAL BEHAVIOR

, 2 : I I I I z I I I I
Ratio of contra-clockwise to clockwise turn- f |

| 1.52|1.400.42|4.00|2.08 4.21/2.23/4.02/0.85 0.93
Range = 1 to 3.60[1 to 4.02 (No. 8) — 1 to 0.42 (No. 3)]

B_ Variation IN DirreRENT Licut INTENSITIES

Light in candle meters ......... ° 0.94 II 39 78 126 | 155 | 217 | 431

Average ratio of contra-clock- ( I I I I I z I I I
wise to clockwise turnings of 4| to to to to to to to to to
the ro worms given in A... | I 1.10 | 1.24 | 1.65 I 0.93 | 1.32 | 1-40 | 1.58

Range = 1 to 0.72 [1 to 1.65 (39 c.m.) — 1 to 0.93 (126 c.m.)]

interesting. A comparison of its activities with those of other
planarians is given in Table XXX, where it will be seen that this
cave worm was considerably more active than any other kind of
worm under observation, both with respect to locomotion and
to the average time required for it to leave a unit circle. Re-
garding the degree of negativeness which it presented, no new
feature appeared, though its average in this point was rather higher
than that of all the other worms studied. However, its degree
of wandering quite exceeded anything shown by planarians which
had been reared in the light.

116 Herbert Eugene Walter

If the relationship of an animal could be determined by behay-
ior alone, there need be no hesitancy in saying that this unidenti-
fed planarian should not be classified under the species Dendro-
coelum lacteum, since in all the criteria mentioned in the foregoing
table it stands at an opposite extreme to Dendroccelum. In
point of fact its behavior more closely resembled that of Phago-
cata gracilis, a species which, according to Dr. A. M. Banta, who
kindly furnished the cave planarian for this study, is common in
the streams in the vicinity of the cave where the latter was found.

TABLE XXX
The behavior of a cave planartan compared with that of planartans accustomed to light.

THE CAVE
OTHER PLANARIANS
PLANARIAN

CRITERIA OF BEHAVIOR ear LE Te
Number Average Average) Maxi- Mini-

Se Range
of trials record record mum mum

Average amount of negativeness, expressed
in the percentage of deviation upon leav-
ing a circle 10 cm. in diameter when
started at right angles to incident light 7o 99-6 91.8 99-9 84.6 15-3
(B delloura omitted)
Average rate in mm. per sec............ 60 2.00 1.203 1.473 | 0.853 0.62
Average seconds required to leave a circle,
LOw CUEING LAN so ctyeeelo,s cose Saat Tees a go 27.8 | 52.2 62.2 39-7 22.5
Average deviation in degrees when pointed
away from the light. (Amount of wan-
ering ee sacberuaees eee cece 46 47 25°.6 | 39°.3 9°54 29°.9

Summary. Average individual behavior constitutes typical
specific behavior. Variations in individual behavior make accu-
rate predictions of responses to stimuli under given conditions,
impossible. The rate of locomotion of the same individuals varies
from day to day even under apparently identical conditions.
Individual variations in the rate of locomotion, in the range
between maximum and minimum rates, and in the percentage of
clockwise turnings, are more variable than the average behavior
in these particulars under different light intensities.

An unidentified cave planarian showed greater activity and
more inclination to wander than any of the other planarians under
observation.

(To be continued)

CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORY OF THE MUSEUM
OF COMPARATIVE ZOOLOGY AT HARVARD COLLEGE. E. L. MARK,

Director. No. 193.

THE REACTIONS OF PLANARIANS TO LIGHT

BY
HERBERT EUGENE WALTER

With Fourteen Ficures

(Concluded)

4 BASIS OF BEHAVIOR

In the sections on Photokinesis and Phototaxis certain con-
ditions of illumination were shown to be variable factors in influ-
encing the movements of planarians. Following this treatment
of the subject an attempt was made under “Kinds of Behavior”’
to classify the effects of light according to the way in which the
responses of planarians become manifested in a generic, a specific
or an individual sense. It now remains to consider the nature of
the factors which cause different individuals to present charac-
teristic differences in behavior. ‘There are at least three ways of
approaching the matter. “These may be roughly indicated as
the point of view of the morphologist, the physiologist, and the

psychologist.
A Morphological Basis of Behavior

The structure and shape of a planarian, its muscular and cil-
lary equipment, together with the kind and distribution of its light-
receiving apparatus, are some of the morphological factors defin-
itely restricting the kind and range of its reactions to light. “These
morphological factors may be grouped into, first, those which
determine the general form of the body and consequently influ-
ence the animal’s activities in a broad sense, and, secondly, those
directly concerned with the reception of the light stimulus, the
photoreceptors.

Tue JourNAL oF ExPEriMENTAL ZOOLOGY, VOL. V, No. 2.

118 Herbert Eugene Walter
a General Form of the Body

A normal, full-grown planarian may be expected to give typ-
ical reactions to any stimulus. Fragments of a planarian, on the
other hand, whether occurring from natural or artificial causes,
would not be expected to behave as perfectly developed worms
do, and observation shows that they do not.

As previously mentioned, Loeb (’94), and later other investi-
gators, established the fact that planarians with eyes and brain
removed are still able to give characteristic reactions to light,
while Lillie (or) found that any fragment capable of regenera-
tion would respond to light.

In all cases of mutilated worms, however, the response to light
is slower and less precise than that exhibited by normal individ-
uals, and therefore different in degree if not in character from
that of the latter. It has been repeatedly observed that worms
mutilated unilaterally perform circus movements regardless of
the light. This seems to be a plain case of morphological limi-
tations on the part of the crippled animal, whereby the cilia and
musculature of one side, on account of injury, are less efficient
than those on the other side. Since it is practically impossible in
nature to select at random a dozen planarians of which at least
one specimen does not show some sort of mutilation, the modi-
fied behavior of morphologically imperfect animals becomes a
factor of considerable importance in any general analysis of
planarian activities.

Again, with regard to the general form of the body, it seems
reasonable to suppose that a mature planarian loaded down with
sexual products, or one gorged with food, must encounter mechan-
ical difficulties in responding to light, so far at least as locomotion
is concerned, which the same animal when unencumbered would
not experience. A few experiments were performed to test this
supposition, in which a comparison of the behavior of large and
small worms was attempted. Pearl (’03, p. 546), it will be recalled,
has shown that in the case of Planaria maculata, a worm II mm.
long travels at a faster rate than one 6 mm. long. This experi-
ment was repeated with a confirmatory result but, as will be seen

The Reactions of Planarians to Light 119

upon examining Table XXXI, the same result did not occur
when Dendroccelum lacteum was used.

TABLE XXXI
The average rate of locomotion in mm. per sec. of 5 small and 5 large individuals of each of four species,

subjected to identical light conditions

Larce SMALL
SPECIES , A

Size Average Size | Average

mm. rate mm. Tate
Dendroccelum lacteum (first trial).......... II 0.695 4 1.01
Dendroccelum lacteum (second trial ........ II 0.74 4 0.77
pha po Cataigraciliss sees sjereieieie.s\cisiele <icieievels 9 1.58 4 Te2T
Planariaimaculatas |. jectccle ccs ce swarm eic}e/-\vin/\s 13 1.57 8 1.37
Planaria gonocephala....................- 10 1.17 5 | 0.98

The worms selected for the experiments detailed in Table XXXI
were carefully chosen as to length and did not vary more than a
millimeter in any case from the size recorded in the table. The
result obtained with Dendroccelum was so unexpected that the
same ten individuals were put aside and tried a week later under
as nearly identical conditions as possible. As will be seen by the
table, the result of the second experiment was in general the same,
though not so pronounced, as that obtained in the first set of trials.
In the cases of Phagocata gracilis, Planaria gonocephala and
Planaria maculata, the larger worms traveled faster than the
smaller ones. Why the factor of size should give a different
result in the case of Dendroccelum lacteum from that common
to the dark-pigmented planarians is by no means clear. It is
conceivable that a planarian with undeveloped sexual organs or
one whose size was reduced through starvation might have a better
ciliary equipment 71 proportion to its mass than a normally adult

animal and that in consequence it could travel faster. “This sup-
; position explains the behavior of Dendroccelum lacteum, but it
does not throw light on that of the other species, of which the
smaller individuals, instead of traveling faster than their larger
associates, moved at a slower rate. It is possible that in the case
of the dark-colored worms reduction in size is accompanied by a

120 Herbert Eugene Walter

corresponding reduction in the photoreceptive elements, which,
according to the experiments of Loeb (’94) and of Parker and
Burnett (’00) seem to be in some degree at least distributed over
the entire body. If this is true, there would result less stimulation
from the light and consequently a slower rate. That Dendro-
ccelum lacteum when reduced in size does not suffer a similar
reduction of its photoreceptive apparatus is probable. ‘The work
of Lillie (’o1), wherein he showed the inability of headless individ-
uals of Dendroccelum lacteum either to regenerate or to respond
to light, suggests that the photoreceptive apparatus of this species
is not scattered over the entire body, but is rather concentrated
anteriorly, in all probability consisting of the eyes only. If this
is true, a reduction in the size of the body would not necessarily
cause a proportionate reduction in the photoreceptors, and,
indeed, the proportion of the light-receiving elements as com-
pared with the mass of the body might increase as the worm
became smaller. In this connection it is interesting to note that
Gissler (’82) pointed out that in the case of Bdelloura candida
increasing size of the body is accompanied by a decrease in the
size of the eyes, and so far may this inverse ratio be carried that
the eyes sometimes disappear entirely inlarge individuals. If there
actually exists some such inverse ratio between the size of the
photoreceptors and the mass of the body in the case of Dendro-
ceelum lacteum, it is easy to see why the smaller worms travel
faster than the larger ones.

By another series of experiments it was found that the smaller

worms of all four species, with the possible exception of Dendro- °

ceelum lacteum, orient with less accuracy than the larger worms
under the same external conditions. In these experiments, as in
the previous ones already described dealing with orientation,
each worm was placed at the center of a circle 10 cm. in diameter
and headed successively toward, away from, and at right angles
in both directions, to the incident light. ‘The average amount of
deviation at the circumference of the circle from the direction in
which the worms were started, reckoned in degrees, gives a crite-
rion of their accuracy in orientation. ‘The averages of behavior

obtained are indicated in Table XXXII.

The Reactions of Planarians to Light I21I

The lessened accuracy in orientation among the smaller worms,
as compared with the larger ones, helps to support the hypothesis
that reduction in size entails proportionate reduction in the
photoreceptive apparatus. The fact that Dendroccelum lacteum
forms an apparent exception to this general rule may also be
taken as evidence that in this case the photoreceptive apparatus
is more localized than in the other worms studied and_ conse-

TABLE XXXII
The average deviation (expressed in degrees), at the circumference of a circle 10 cm. in diameter, of
large and small worms, each lot consisting of 5 individuals. Each worm was headed successively
toward, away from and at right angles in both directions to incident light. The actual sizes of

the worms were the same as in Table XX XI

DENDROC@LUM LACTEUM PLANARIA |

Phagocata| Total

S1zE OF WORMS s q | x
Sion gono- | gracilis average

First trial tale | Average | maculata eeerate
|
Large, degrees... 67 { 7° | 68.5 57 64 63 } 63
|

Small, degrees... 57 } 85.5 68.5 61 69 72 67.4

quently does not suffer a proportionate decrease when the size of
the body becomes less. It is furthermore quite possible that a
sexually mature planarian may on that account behave differently
in light than an immature one. For instance, Yerkes (’03) states
that in the case of the hydromedusa Gonionemus murbachii, the
sexually mature individuals are the ones most sensitive to light,
and Schouteden (’02) found the young of Daphnia positive, while
- the adults were negative to light.

Finally, Harper (’05) has shown that in the case of the earth-
worm the degree of sensitivity to light depends upon the degree
of contraction or expansion of the body, since the photoreceptor
cells—which in the earthworm lie interstitially at the bases of the
epithelial cells—are more exposed to stimulation when the worm
is expanded and conversely more shielded when it is contracted.
It is more than likely that planarians offer a parallel instance
and that their comparative indifference to light stimulation when
in the relaxed resting position is due to the fact that then they pre-
sent a more rounded contour and consequently their photoreceptors

122 Herbert Eugene Walter

are more deeply buried from the light than when they are in the
expanded position assumed while gliding.

b_ Photoreceptors

What is the photoreceptive apparatus of the planarian? Is it
made up of the eyes only, or partly of nerve-endings or of some
special morphological elements homologous perhaps to the pho-
toreceptor cells in the integument of the earthworm as described
by Hesse (’96). Or does the central nervous system, the ciliary
apparatus, or the musculature receive the stimulus directly with-
out the mediation of special sense organs ?

Although these questions were not made the subject of parti-
cular investigation in the present study of planarian behavior,
certain facts incidentally appear from the observations made for
other purposes which bear directly upon these inquiries and may
serve as a basis for a brief discussion of the nature and location
of the photoreceptive apparatus of planarians. ‘The presence of
eyes in the anterior part of the body, together with the wigwag
movements which often take place in the same region when a
variation occurs in the light conditions, point directly to the con-
clusion that the anterior end of the worm is more responsive to
light than the posterior end. ‘Uhe fact that many planarians con-
tinue to react to light with considerable definiteness after the
whole anterior end of the body is removed, indicates that this
region does not necessarily contain the entire photoreceptive
apparatus. Decapitated individuals of the species Dendro-
ceelum lacteum, it should be noted, seem to be exceptional in this
respect since, according to Lillie, they fail to react to light.

In further support of the supposition that the anterior end of
the planarian is the portion most sensitive to light it was found
that the skioptic response of Bdelloura candida is confined not
only to the anterior end but particularly to the region directly
including the eyes. Observations repeatedly showed that if
Bdelloura was allowed to come to rest in a field illuminated from
above only, a sharp narrow shadow thrown across its body pro-
duced no visible response unless the shadow included the eyes.

The Reactions of Planarians to Light 123

The moment, however, that the eyes were in shadow the worm
would elongate and frequently begin strikingly active movements.

It has already been shown (Table XIV, p. 77) that all the
different species of planarians upon which experiments were made,
traveled at a faster rate when they were started with the ante-
rior end pointed toward directive light than when away from it.
A reason may be offered for this characteristic increase in rate on
the ground that the anterior end was plainly subjected to stronger
stimulation when directed toward the light than when pointed
away from the source of the stimulus. In the latter instance it
was not only turned away from the source of the stimulus but
was shielded also from the light to a considerable extent by the
shadow formed by the posterior part of its own body.

Again, when a small beam of sunlight passing through a_ pin-
hole in an opaque screen was directed locally to different parts of
a gliding Planaria maculata, it was found that tropic response
would occur in case one side of the anterior end was illuminated,
and that it was not necessary for the eye itself to be included in
the illuminated area to obtain such responses. However, when
the middle of the body or the posterior end was similarly stimu-
lated the worm could not be made to turn.

From the foregoing observations it seems probable that the
photoreceptive apparatus of planarians is mainly but not exclu-
sively located in the anterior end of the body and that considerable
specific or generic difference may exist with respect to the extent
of the distribution of additional light-receiving organs over other
parts of the body. It is interesting to note in passing that Gamble
and Keeble (’03) found that in the case of the green rhabdoccele
Convoluta roscoffensis the sensitiveness to light was at the ante-
rior end of the body only.

Concerning the relative sensitiveness to light of the dorsal and
ventral surfaces of planarians, a set of experiments was performed
on Planaria gonocephala in which the results show an absence
of any marked differentiation in this regard. It is well known
that in the matter of response to a thigmotactic stimulus the dor-
sal and ventral surfaces of a planarian show a very striking differ-
ence. Indeed, the dorsal surface is negatively thigmotactic to

124 Herbert Eugene Walter

such a degree that it is practically impossible to make a worm
remain with its dorsal surface in contact with any surface, while
its ventral surface is just as strongly positive in its thigmotaxis.
In contrasting the receptivity of these two surfaces to light
stimulation a field of two adjacent intensities, similar to that used
in the experiments on abrupt spacial changes in light intensity
(Fig. 3, p. 65), was arranged in such a way that, in the first instance
the source of the two lights was below, and in the second above,
the field in which the worms were placed. ‘The intensities of the
light in each case were approximately 66 and 33 c.m. By this
means the responses of the worms could be tabulated as they
glided from one intensity of light to another and those given when
the light impinged on the dorsal surface compared with similar
responses made when the light struck directly on the ventral sur-
face. It will be seen in Table XX XIII that the results do not indi-
cate any particular difference for the dorsal and ventral surfaces
with respect to the distribution of the photoreceptors. ‘This con-
dition of affairs, however, may be largely due to the translucency
of the planarian’s body, which would render light-receiving organs
accessible from whatever direction the light primarily comes.

TABLE XXXIII
A comparison of responses made by Planaria gonocephala to a change in light intensity, tabulated with
reference to the source of the light and its relative degree of stimulation upon the dorsal and ventral

surfaces of the worm respectively

Number of | Percentage of Perce eeok

ition of light wigha
Position of lig’ observations | total responses | Bee
movements
|
IOI | 5° Gre
156 | 53 36

An exact determination of photoreceptors other than the eyes
was not made. Both Iijima (’84, p. 438) and Carriére (’82, p. 167)
in their histological researches upon planarians found “‘Neben-
augen’’ frequent and these occasional accessory eyes have also
been described by Janinchen (’96, p. 259). Such structures may

The Reactions of Planarians to Light 125

possibly be interpreted as the connecting link between undifferen-
tiated light-receiving organs and the normal eyes of the planarians.

The part that pigment plays in the reception of light is not as
yet clearly defined. It is not probable that pigment in itself con-
stitutes a photoreceptor, though it is usually found associated
with sensory cells which are directly concerned with light reception.
That it is not an essential factor of a photoreceptor is evident,
inasmuch as it is absent from the eyes of albino animals. The
secondary role of pigment in the reception of light by organisms is
admirably pointed out and discussed by Beer (’01).

The presence of pigment in a planarian may, however, modify
the animal’s response to light stimulation by shielding the sen-
sory cells from light, and since its distribution in general is near
the exterior, it may afford some clue to the relative receptivity
of internally and externally situated photoreceptors. In other
words, if pigmented and non-pigmented worms, for example, ex-
hibited the same behavior in light, it might reasonably be assumed
that the photoreceptors were not located internally, since they
would be partially shielded from light in the case of the pigmented
forms and consequently would give rise to a different response.

It is of interest, therefore, to contrast the behavior of dark-
pigmented worms with those in which the dark pigment is absent
except in the eyes. ‘This is done in Table XXXIV, but it by no
means follows that the contrasts there given between the behavior
of dark and light worms are due to the presence of dark pigment
in the one case and its absence in the other. Other factors than
pigment may very probably have been influential in bringing
about variations in the light reactions tabulated. Furthermore,
it is inaccurate to refer to a white worm as being non-pigmented,
since in that case it would be entirely transparent. ‘The question,
then, so far as planarians are concerned, is confined not to differ-
ences between pigmented and non-pigmented but to differences
between dark-pigmented and light-pigmented forms.

It will be seen from Table XXXIV that when subjected to light
stimulation dark-pigmented worms in general show more activity
than light-pigmented forms. A single exception to this rule
occurred in the case of the cave planarian experimented upon.

126 Herbert Eugene Walter

This marked difference in behavior might possibly be explained
on the hypothesis that the direct effect of light on the deeper lying
nervous system is inhibitive; that is, so excessive as to produce a
sort of light rigor. ‘Thus the more the central nervous system is
shielded from light by pigment the less the inhibitive effect becomes
apparent. Certain it is that Bdelloura candida, which has dark

TABLE XXXIV

The behavior of dark-pigmented worms contrasted with that of worms not possessing dark pigment dis-
tributed over the body. The number of observations in each case ts not given since the details of this

table have already appeared elsewhere

Dark PIGMENTED LiGuT PIGMENTED

s eae} &,

a S

S| & le as|A =< los

Duration of movement in a typ-

ical set of experiments, minutes) 47 60+ | 60+ 56+ 18 15 | 6s
Percentage of orientation to light

upon coming torest.......... 41 41 30 30
Wigwag responses at the critical

line separating two intensities

of non-directive light, per cent 39 39 8.5 | 8.5
Average number of seconds re-

quired to escape from a circle |

10 cm. in diameter ........... 48.8 | 59.6 | 39-6 | 49.3 | 62.2 27.6 | 44.9
Precision of response
Deviation in degrees upon emerg-

ing from a circle 10 cm. in dia-

meter when headed away from |

the ightsrcyarcievaa iterate s(cte(=teieiets= ay Hae D ey 29.1 26.3 10.1 11.4 | 10.7
Rate of locomotion in mm. per
SECanaciteccsee el tteretersiace 1.47 | 1.075| 1.395| 1.28 | 0.85 0.85

pigment in its eyes only, may be brought to a standstill very read-
ily by means of light stimulation. With the exception of the eyes
it may be possible that the photoreceptive apparatus is not differ-
entiated to such an extent that it could fairly be said that any part
of the translucent planarian body is entirely free from the direct
stimulation of light. The relation of pigment to light reactions

The Reactions of Planarians to Light 127

is, however, by no means settled in the foregoing observations.
This matter should be finally tested by comparisons in the behay-
ior of different individuals of the same species showing variation
in pigmentation or of identical individuals at different times when
their phases of pigmentation are unlike, rather than upon indi-
viduals of different species.

It has proven impossible to include such a consideration in the
present paper, but the preliminary steps toward attempting an
analysis of the function of pigment with reference to light reactions
have already been made and it is expected that a discussion of
this phase of planarian behavior will be presented later. It may
be stated here that when Planaria maculata is fed with a drop
of human blood, a decided increase in pigmentation makes its
appearance within a few days, due probably to the oxidation of
the hemoglobin in the blood corpuscles with which the planarians
have Become gorged. ‘This single observation suggests an experi-
mental means oe controlling che amount of pigment in a single
individual and it may reasonably be supposed that tests of eae
ior before and after excessive pigmentation will contribute direct
evidence upon the part played by pigment in reactions to light.

Summary. Mutilated planarians in general respond to light
with less accuracy than normal individuals.

Small worms move more slowly than large ones in the case of
those species whose photoreceptive apparatus 1s not solely confined
to the anterior end of the body. In the case of Dendroccelum
lacteum, whose photoreceptive apparatus is relatively greater in
small individuals than in large, the rate of incomouonet faster
among the smaller than among the larger.

Small worms orient with less accuracy than large ones. Pla-
narians in the relaxed, resting position are less responsive to light
than when they are stretched out in the act of gliding, a result
probably of the greater exposure of the photoreceptors to light in
the latter instance.

The anterior end of the body 1s the chief photoreceptive region
and in certain worms, such as Dendroccelum lacteum and Bdel-
loura candida, the anterior end is apparently the exclusive seat of
this function.

128 Herbert Eugene Walter

No marked difference in response to light is shown between
worms stimulated on the ventral surface and those equally stim-
ulated on the dorsal surface.

Aside from the eyes, which form at least a part of the photo-
receptive apparatus, no definite light-receiving organs were recog-
nized.

Planarians possessing dark-colored pigment distributed over
the body show in general greater activity when subjected to light
than forms in which there is no dark pigment except in the eyes.

The central nervous system, as well as the more exterior parts
of the planarian, may possibly be stimulated directly by such light
as passes through the translucent body.

B The Phystological Basis of Behavior

The continually changing adjustment in any organism between
the incoming and the outgoing energy gives rise to varying phases
of metabolic balance, which may be designated as different “ physi-
ological states.” Such physiological states form a noticeable fac-
tor in the behavior of any animal, a fact towhich Jennings (’o4b,
p- 109) in particular has called attention.

That the difference between such states is great may be readily
demonstrated. A planarian’s response to directive light when it
is in a relaxed, quiescent condition is plainly different from that
exhibited after it has been vigorously disturbed by a brush. In
fact, it is extremely difficult to get two animals that are in precisely
the same physiological condition, or the same animal in precisely
the same state at two different times, since the exact adjustment
of physiological states is too. delicate a matter to be controlled by
the present gross experimental methods.

The attempt is ordinarily made to eliminate from experiments,
so far as possible, the disturbing element of changing physiological
conditions, that is, to keep constant all the factors except the one
which is being subjected to test, and those results are counted as
most successful in which such disturbance is reduced to a minimum.

It is the purpose of this section first, to give a possible classifica-
tion of the different physiological states in which a planarian may

The Reactions of Planarians to Light 129

be, and, secondly, to pass briefly in review some of the many ways
in which light may change the physiological state of such a worm.

a Classification of Physiological States

It is by no means easy to define even a simple physiological state,
since the subtle changes form a continuous series of conditions
which pass imperceptibly into each other.

An arbitrary classification for convenience may, however, be
made as follows:

1 Relaxation, or rest.

2 Slight activity, without locomotion.

3. Normal activity.

4 Violent activity.

5 Rigor.

6 Exhaustion.

In the first of these states there is a minimum expenditure of
energy caused by the ebb of the katabolic processes.

The second and fourth states indicate what are often referred
to as conditions of low and high “tonus,” but as this term has a
technical significance with reference to muscle reactions, it will not
be used in this classification. Thethird state, thatof normal activ-
ity, is the average condition; it is the most desirable one to main-
tain in testing the animal’s responses to different stimuli. By
rigor is understood a state wherein there may be an excessive outgo
of energy, but unaccompanied by movement, while under exhaus-
tion is cneleded the condition when energy is not being released
because there is none to release.

That excessive or continuous light stimulation may go beyond
the point producing rigor or exhaustion and may actually cause
death, has been repeatedly proven in the case of bacteria by a
long line of observers. The inhibitive effect of excessive light
upon other organisms than bacteria has been pointed out by
Berger (00) with reference to Cubomeduse; by Pearl and Cole

® Tyndall (’78), Downes and Blunt (’77, ’78), Jamieson (82), Duclaux (’85a,’85b, ’g0), Arloing
(’87a,’87b), Roux (’87), Dandrieu (’88), Raum (’89), Pansini (’89), Janowski (’90), Buchner (’92) ,
and Ward (94).

130 Herbert Eugene Walter

(02) in the case of various infusoria as well as Hydra, Hyallela,
Clepsine, Stichostema and Physa; by Yerkes (’03) for Goni-
onemus and by Carpenter (’05) for Drosophila.

b Changes in Physiological States Induced by Light

A variety of stimuli besides light may cause an animal to pass
from one physiological state to another. For: example, the sense
of phototaxis was reversed through mechanical stimulation by
Towle (’00) in Cypridopsis and by Holmes (’or, ’o5b) in Orches-
tia and Ranatra.

The following typical illustration of the manner in which
changes from one physiological state to another succeed each other
is offered as a basisof comparisonwith the responses tolight itself,
which are about to be described. In the absence of mechanical
stimuli a planarian may be in a state of relaxation. Very gentle
mechanical stimulation causes the worm to lift its anterior end
and move it cautiously about, bringing the animal into a state of
slight activity without locomotion. If, now, the mechanical stim-
ulus is prolonged or increased in intensity, enough energy is
released to put the animal into gliding locomotion, when it may be
fairly said to have passed into the state of normal activity. Pro-
vided the stimulation is made still more pronounced, the worm
can next be forced to forsake gliding for crawling or humping, so
passing into the state of violent activity. Further, it is possible by
vigorous shaking to throw the worm, temporarily at least, into a
condition of inactivity through excessive stimulation, during which
the animal would remain quiet, not because it is failing to release
any energy, but because it is unable for the time to set free its
energy in the form of locomotion. In other words, it is in the
state of rigor. Last of all, if mechanical stimulation is repeatedly
applied a condition of exhaustion will appear when the worm has
no more available energy and so is unable to move at all.

Effect of Different Intensities. As already pointed out, no inten-
sity either of directive or non-directive light was found sufficient
to change the condition of normal gliding into crawling.

The Reactions of Planartans to Light 131

Moreover, light of any intensity or direction frequently proved
ineffective in arousing a quiescent worm into any state of apparent
activity, particularly if the worm had but recently passed into the
state of rest after a prolonged period of exercise.

Effect of Excessive Light. In the experiments with non-direc-
tive light it appeared that Planaria gonocephala, when subjected
to an intensity of 431 c.m., showed somewhat less activity than at
lower intensities, both with respect to rate of locomotion (Table
III, p. 57) and to the number of turnings made (Table VI, p. 59);
yet, so high a degree of intensity of the light stimulus was appar-
ently not sufficient to cause a change into the physiological state
of light rigor. It was comparatively easy, on the other hand, to
transform Bdelloura candida by means of excessive light from
the state of normal activity into that of light rigor.

Effect of Sudden Change in Light Conditions. A sudden
change in light intensity either by increase or decrease is more effec-
tive in producing a new physiological state than an equal grad-
ual change. ‘The sudden withdrawal of the lamp to a consider-
able distance, for example, is usually sufficient to throw a worm
from a normal state into violent activity, that is, from a gliding
movement into a disturbed state in which the anterior end is
waved actively about. But if the light is gradually withdrawn
the same distance the worm will usually not pass into a different
physiological condition.

The sudden introduction of complete darkness was never
found sufficient to reduce an active worm more than temporarily
to the resting position. Sudden dark might temporarily halt a
moving worm, but it would not cause it to come to rest and assume
the relaxed contour. In Bdelloura candida sudden dark, instead
of checking the animal’s movements, threw it into violent activity.

Effect of Continued Exposure to Light. Continuous exposure
to light results in fatigue, which finally causes planarians to change
from the state of normal activity to that of relaxation. ‘The tend-
ency toward such a change is shown in Table XXXV, where the
responses of a number of worms newly subjected to light stimu-
lation are contrasted with the responses made by the same worms
after they had been moving about for several hours in the light.

132 Herbert Eugene Walter

The fresh worms show more activity than the fatigued worms do.
Otherwise expressed, the worms have a tendency to change into
a lowered physiological state upon continued exposure to light.

TABLE XXXV

Fatigue effects due to continuous exposure to non-directive lights forming adjacent fields of different

intensities, as shown in the behavior of Phagocata gracilis

Ratio oF THE TWo

-96: 13-4501 Average
INTENSITIES iene ae 3-45 3
Percentage of the re- Going Going Going | Going Going | Going
sponses at the crit-| into into FF into into 4 into into a
ical line separating greater lesser & | greater | lesser Pe greater lesser is
= iS ' 0 f 2 &
the two intensities intensity intensity 2 intensity intensity 2% intensity intensity oa
Fresh worms........ | 10.5 | 21 16 45-5 47-5 | 46.5 | 28 34-4 | 31+
Fatigued worms.... 2.5 9-5 32-5 33-5 | 33 17.05 21.5 | 19.5

It may be incidentally noted in Table XXXYV that, as has already
been pointed out in another connection, the percentage of responses
is greater when the contrast between the light intensities is greater,
and that both fresh and fatigued worms respond oftener upon
going into the lesser intensity than when going into the greater
intensity.

The time required for a worm placed in directive light to come
to rest; that is, to run the gamut from the state of normal activity
to that of rest, becomes gradually shorter with continuous expo-
sure. As fatigue increases the worm shifts down the scale of physi-
ological states in less time than when freshly subjected to directive
liohe A specific case of this kind has already been described in
the paragraph on “duration of movement”’ (p. 105); where in 39
consecutive trials the change from normal activity to relaxation
was first made in 18 minutes, but the thirty-ninth time in Ir}
minutes, while the fortieth time even mechanical stimulus failed
to arouse the exhausted worm from the resting position.

Effect of Previous Exposure to Dark. Worms kept several
hours in complete darkness make a larger percentage of re-
sponses to changes in their light environment than those which
previous to experimentation have been several hours in light.
Individuals removed from the stimulus of light for any consider-

The Reactions of Planarians to Light 133

able time are more responsive when subjected to it, for the reason
that they are in a physiological state farther removed from fatigue
than those worms which have remained a long period in the light.

This point is brought out in Table XXXVI.

TABLE XXXVI

Percentage of reactions of two worms, Planaria gonocephala,to a sudden change in light intensity both
when previously kept several hours in the dark and also when previously exposed for several

hours to light

Percentage of responses Number of observations
After several hours in the light........... 54 100

After 48 hours in the dark............... 66 100

Summary. Physiological states grade imperceptibly into each
other, but may be tentatively divided into: 1, relaxation; 2, slight
activity; 3, normal activity; 4, violent activity; 5, rigor; 6, exhaus-
tion.

Various stimuli besides light may induce a change from one
physiological state to another.

No light intensity lower than 431 c.m. is sufficient to throw a
worm into a higher state than that expressing normal activity,
nor is the absence of light sufficient to bring a planarian to rest.

Excessive light intensity shows a tendency to carry Planaria gono-
cephala from a state of normal activity to one of rigor. Bdelloura
candida is easily changed into a condition of rigor by light.

A sudden change of light intensity acts more immediately than
a gradual change in causing planarians to pass from one physio-
logical state to another.

Continuous exposure to light induces fatigue, finally resulting
in the passage of the worm into a state of continuous relaxation,
in which condition it becomes practically indifferent to light.
Repeated trials of the time required in constant light to come to
rest show that a progressively shorter interval occurs between the
state of normal activity and that of relaxation until a point of
complete inactivity is reached, the worm finally remaining in the
latter state for a prolonged period.

134 Herbert Eugene Walter

Planarians kept for some time in darkness pass into a state in
which they are more’responsive to light than individuals exposed
for a similar length of time to light.

G Psychological Basis of Behavior

Among the first questions that naturally arise concerning the
behavior of planarians in light are those which approach the mat-
ter from a psychological point of view. How much can planarians
actually see, and can they, by repeated experience, “learn” to
adapt themselves to changes in the light surrounding them ¢

To this kind of inquiry it is most difficult to give a satisfactory
answer, for the reason that it is impossible to go beyond conjecture
and inference in judging what any animal, aside from man, can
see or know or experience. It is only possible to state, in more
or less definite terms, the responses which animals make to light,
since it is beyond man’s power ever to experience how animals
“feel” under any circumstances.

a. How Much Can Planarians See?

Broadly speaking it may be said that planarians can distinguish
light from darkness. The experiments described on pp. 84,
et seq., relating to planarians placed ijn aquaria so surrounded by
backgrounds as to produce regions of different light intensity,
point to this conclusion, since when subjected to such differential
environments the worms come to rest in the darkened areas.

Again, the numerous responses made at the critical line separa-
ting two light intensities may be regarded as evidence of some
power of discrimination on the part of the worm between dif-
erent intensities of light.

It is probable, furthermore, that planarians can distinguish a
moving object when that object is of sufficient size and contrasts
with its surroundings in its degree of illumination, for the reason
that a moving object from which light is reflected, means the same
to a worm coming into the vic:nity of the object as any other change
in the direction of light, such as might be caused by moving a

The Reactions of Planartans to Light 135

lamp from one position to another. ‘To changes in directive light
planarians are known to respond very definitely, and consequently
they may be said to distinguish the motions of objects.

With regard to true seeing, however, in the sense of distinguish-
ing the forms of objects, it is safe to assume that planarians have
almost no power whatever, since their eyes are optically unable to
form images even if the central nervous system were highly enough
developed to interpret images when formed. In the case, there-
fore, of Planaria alpina, which, according to Collin (91, p. 180),
“shuns” Planaria gonocephala when the latter has been put into
the same aquarium with it, seeking “‘strenuously to escape” from
its larger relative, the conclusion does not necessarily follow that
P. alpina sees an enemy and experiences the sensation of fear.
As previously pointed out (p.95), the whole matter is probably
explainable on the basis of negative chemotaxis alone. ‘To attri-
bute fear, therefore, or any other similar complex sensation, to
an organism whose responses are so plainly of a simple reflex
nature, is to go quite beyond the evidence.

In the performance of the two great life processes of nutrition
and reproduction, light is apparently in no way a direct aid to pla-
narians, since they thrive in situations from which light is entirely
excluded, as in caves, and since they habitually frequent places
where this factor is reduced to a minimum. Light cannot, then,
be regarded as a directly essential factor in the life of planarians.

That light is not essential to the activity of protoplasm has more
than once been demonstrated. Engelmann (’79), for example,
showed that the streaming protoplasm of plant cells occurs nor-
mally in darkness, while Maupas (’87) found ciliates multiplying
as rapidly in the dark as in the light.

b~ Are Planarians Able to “Learn” ?

With regard to the ability of these worms to acquire upon repe-
tition an abbreviated form of response; that is, to “learn,” a few
suggestions may be drawn from experiments already described
in other connections.

It will be remembered (p. 93) that when a small aquarium

136 Herbert Eugene Walter

was delicately mounted upon a turntable, such as is used in “‘ring-
ing’’ microscopic slides, a very slight rotation was sufficient to
bring to a halt momentarily a gliding worm in this aquarium.
It was possible to control this momentary response to such a
point of nicety that the anterior end of the worm could be made
to halt for an instant without interfering with the onward loco-
motion of the posterior end. If this slight rotation was repeated
at intervals of a second it was found that the worm under obser-
vation halted with less and less certainty, until after a dozen or
more trials it continued to glide on without halting at all. In
ordinary phraseology the worm had learned by experience not
to be alarmed by a sudden mechanical shock. ‘The lesson, how-
ever, was always very soon forgotten, for after an interval of less
than a minute, during which the aquarium remained stationary,
the worm responded exactly as it did at first, whenever a slight
rotation was made. In a similar way the skioptic response of
Bdelloura candida became less pronounced upon repetition, until
it was possible to throw a shadow upon the animal without
obtaining any response at all.

Again, when worms were placed in a field of non-directive light,
parts of which were of two different intensities, the number of
wigwag responses made at the critical line separating the two
intensities grew less after the animals had repeatedly crossed the
line. At first the new condition of sharply contrasted light
intensities in the worm’s field of locomotion called out a large
percentage of wigwag responses. Later, however, by repeated
experiences the worm became familiar with this feature of its
environment and made fewer wigwag motions. A definite
instance of such a decrease in response is given in Table XX XVII.

TABLE XXXVII

Responses of Planaria gonocephala on crossing the line separating two intensities of non-directive light

Wigwag movements No response Percentage of response
First 25 crossings....... 21 4 84
Second 25 crossings..... 19 6 76
Third 25 crossings...... 12 13 48

Fourth 25 crossings ..... | 8 17 32

The Reactions of Planarians to Light 137

It will be seen that when Planaria gonocephala was first intro-
duced into a field of contrasted intensities, it made the wigwag
response at the critical line marking a change of light intensity, in
84 per cent of the first 25 crossings, while during the second, third
and fourth sets of 25 crossings, the per cents uniformly decreased
until at the fourth 25 crossings the number of wigwag responses
fell to 32 per cent. It may be objected that the instances thus far
cited in this section find a more reasonable explanation upon the
hypothesis of fatigue, but the same surely cannot be said of the fol-
lowing case.

It was found that Planaria maculata oriented itself to directive
light at successively shorter intervals when the position of the
light was suddenly changed. To produce such a series of re-
sponses there was placed in the dark room a shallow aquarium
with an electric lamp at either end, under the control of the right
and left hand, respectively, of the experimenter. A planarian was
placed in the middle of the aquarium and the right-hand light
turned on. As soon as the worm was fairly oriented to this light
and gliding away from it, the right-hand light was turned off and
at the same instant the left-hand light turned on. The time in
seconds required for the worm to orient to the new light; that is,
to turn 180° and begin to glide away, was recorded. On p. 89
a typical series of records of such responses is given, in which the
number of seconds required for re-orientation when the source of
light was reversed, varied from 260 seconds, at first, irregularly
down to 35 upon the sixteenth trial. It will be seen from this series
that the worm acquired by experience some degree of facility in
adapting itself to certain variations in its environment which it
would never be liable to encounter in nature, and that this adapta-
tion cannot be explained as due to fatigue. Davenport and Cannon
(97, p- 32) found similarly that “‘ Daphnias respond more quickly
and accurately to light after having made several trips to it.”

It is quite certain, however, that any educative attainment
which a planarian may experience, or which a planarian may
acquire, is exceedingly evanescent and also that there is no evi-
dence that the worm emerges from reflex behavior into responses
connected with consciousness.

138 Herbert Eugene Walter

Summary. The existence of feeling or consciousness among
planarians is a matter of pure conjecture.

From their responses it may be inferred that they are able to
distinguish dark from light, as well as objects in motion, but it is
not clear that they can distinguish the forms of objects.

The knowledge which planarians have of objects in their imme-
diate environment, such as food, enemies, etc., depends largely
upon chemical and tactile means. ‘They are, therefore, as well
able to go through the entire range of their activities in the dark
as in the light.

Upon repetition planarians may in some instances become accus-
tomed to, or acquire greater facility in, responding to stimuli, but
this result of experience is almost instantly lost, so that it is doubt-
ful whether these animals possess more than the merest rudi-
ments of the primary criterion of consciousness, namely, the ability
to learn.

VI GENERAL CONCLUSIONS

Probably the questions which have occupied the greatest share
of attention throughout the literature dealing with the reactions
of organisms to light, are the following:

1 Is the direction or the intensity of light of more importance
in orientation ?

2 Which theory best explains orientation and phototaxis, the
theory of trial and error or that of the tropisms ?

3. How far is behavior with respect to light, adaptive ?

I DIRECTION OR INTENSITY

Before the part played in the behavior of planarians by either
the direction or the intensity of light can properly be discussed,
it will be necessary to present a brief historical résumé of certain
general conclusions reached by investigators along this line.

A Historical
Cohn (’53), Strasburger (’78) and Loeb (’g0, ’93a) attributed

the directive effect of light to the action of the rays. In a later

The Reactions of Planarians to Light 139

paper Cohn (64) abandoned his first position and came to regard
intensity as the important element in light, a position also main-
tained by Famintzin (67), Engelmann (’83), Oltmanns (’92),
Verworn (’or) and even by Loeb (’93b) in the case of Planaria
torva, which he found came to rest in accordance with the intensity,
and regardless of the direction, of the light. Davenport and Can-
non (’97) modified this point of view by attempting to show that
direction and intensity may each operate independently, producing,
respectively, “phototaxis”’ and “photopathy.”’ Holt and Lee (’or)
followed with an excellent summary of the whole controversy,
emphatically maintaining, in opposition to Davenport and Can-
non, that intensity alone is the only possible operative factor in
light stimulation and that direction of the rays has no effect what-
soever except in determining a greater intensity of light with refer-
ence to one part of an organism as compared with other parts.

Among more recent investigations Holmes (’03), experimenting
with the same organism that fled Oltmanns to ascribe the greater
importance to intensity, namely, Volvox, declares himself in favor
of direction, while Zeleny (05), on the other hand, gives an
instance of Serpulid larve going both toward the source of the
light and away from it; that is, moving regardless of direction, in
order to arrive in regions of increased intensity.

Carpenter (’05) found that the pomace fly, Ampelophila droso-
phila, will orient to the direction of light after it has first been
sufhciently aroused by the intensity of the light, while both Yerkes
(99) and Towle (’oo) maintain that direction and intensity are by
no means mutually exclusive, and that each may play a part simul-
taneously in determining the behavior of an organism.

Lastly, it has been made clear by Parker (’03) that, besides
direction and intensity of light, the size of the source of illumina-
tion may determine the orientation. ‘This theory explains why
butterflies alight upon a patch of reflected sunlight which produces
a large but faint retinal image instead of flying toward the sun
itself, which forms only a small but intense retinal image. In the
case of planarians, however, this phase of light stimulation is not
operative, since the eyes of these animals are incapable of forming
retinal images.

140 Herbert Eugene Walter
B Conclusions with Reference to Planarians

The behavior of planarians may in general be more satisfac-
torily explained by regarding, with Loeb, the intensity rather than
the direction of the light as the principal operative factor in light
reactions. At the same time there is much evidence that the inten-
sity utilized by the organism, is intimately associated with, and
powerfully modified by the direction of the light. As a basis for
these conclusions the following points will be considered. First,
the distinction between direction and intensity; secondly, the way
in which directive light modifies the intensity with reference to
planarians; thirdly, the action of intensity without the modifying
effect of direction, and finally, modifying effects of factors other
than light.

a. The Distinction Between Direction and Intensity

Theoretically it is plain that light per se with respect to any fixed
point, may be regarded in two distinct aspects, namely, that of
intensity and that of direction. ‘The intensity of light under ordi-
nary circumstances varies inversely as the square of the distance
and is independent of the position of the source of light. That
is to say, at any points equidistant from its source, light has the
same intensity, but the more remote the less is the intensity at
any given point. ‘The direction of light, on the contrary, is depend-
ent solely upon the position of the source of the light and in no
way upon the distance. When intensity and direction are con-
sidered with reference not to a fixed point but to an organism
presenting three dimensions and made up of differentiated pro-
toplasm, the basis of light relations becomes more complex.
Light cannot here be treated as a phenomenon per se but must be
considered in relation to a differentiating organism.

It is true that intensity in the case of the organism, as in the case
of a fixed point, varies with the distance from the source of the
light. A decided difference, however, appears in the case of the
organism inasmuch as, owing to its structure, the intensity received
by it varies also in accordance with the position of the light. ‘This

The Reactions of Planarians to Light I4I

second form of variation in intensity is directly due to the fact
that the organism has a solid form and is not homogeneously photo-
receptive.

The direction of light with reference to the organism, presenting
as the latter does a structurally diversified form, is influential only
as regards the position of the source of light, just as in the case of
a fixed point.

Any change in the position of the source results, then, in a
redistribution of the intensities falling upon the organism, so that
again the intensity received varies in accordance with the position
of the light.

It is this factor of posztion in light that has been termed the direc-
tive influence of light and it is seen to be due to variations in
the intensity of light with reference to the organism, and not to
any peculiar property of light iself. By “‘non-directive light,”
on the other hand, is understood those conditions which secure
for the organism equalized or symmetrical intensity with respect
to the parts stimulated. If this interpretation is correct there can
be no response, strictly speaking, to the direction of light exclu-
sive of intensity although the factor of intensity may be continually
modified by that of direction in the light relations of organisms.

b The Modifying Influence of Direction

It is undeniable that the planarians experimented upon exhib-
ited without exception a definite characteristic phototaxis, that
is to say, they habitually go either toward or away from the source
of light according as they are respectively positive or negative.
In analyzing this phototaxis it seemed desirable to eliminate so
far as possible the factor of intensity, but the attempt to do this
was only partially successful owing to physical limitations. A
step was made, however, toward subjecting worms to directive
light without at the same time exposing them to a variation in
intensity by inserting a biconvex lens between the source of the
illumination and the aquarium, thus making the diverging rays
of light parallel throughout their course in the aquarium. By this

“means was formed a field equal in its amount of illumination at

142 Herbert Eugene Walter

the two ends of the aquarium, the one opposite and the one next
to the source of light, with the exception that there was a slight
difference at the two ends due to the fact that light in its passage
through water is partially absorbed. But modification of light
in any degree results in producing less intensity at the farther end
of the aquarium, though this difference is less pronounced when
a lens is employed. ‘Therefore, although worms placed in this
apparatus went with considerable precision in the direction of the
propagation of the light, there is no certainty that their behavior
was not due simply to differences in intensity. Worms which are
thus apparently traveling directly in accordance with the direc-
tion of the light, are meantime being subjected to different inten-
sities at the anterior and posterior ends of the body, for the reason
that the anterior end is more or less shadowed by the rest of the
body, since the latter cuts out a certain portion of the light received
at the posterior end.

That direction of light is a factor by no means to be disregarded,
even if it cannot be proven to be the immediate cause of phototaxis,
is apparent when it is recalled that slight changes in direction
call out corresponding changes in the course of the gliding pla-
narian, whereas considerable changes in intensity when the direc-
tion remains constant and particularly when such changes are
gradually made, may fail entirely to produce corresponding
changes in the worm’s behavior. ‘This is due to the fact that slight
changes in direction may cause considerable changes in the asym-
metry of illumination. When a worm, for example, is receiving
horizontal light from behind, its head is more or less in shadow,
the sides of its body being at the same time equally illuminated.
The moment the light is shifted in even a small degree to one side,
one entire side of the animal may receive an increase of illu-
mination and the opposite side be thrown into shadow. Thus a
slight change in position initiates a fundamental change in the
distribution of intensity over the planarian’s body.

c Instances of Behavior Due to Intensity Alone

The effect of intensity as a separate factor from the directive
influence of light is clearly demonstrable in certain phases of

The Reactions of Planarians to Light 143

light reactions. Yo isolate intensity by excluding the possibility
of directive light; that is, to secure equalized intensity with refer-
ence to the organism, is not difficult and the manner in which this
was done, with non-directive light falling upon a horizontal field
from above, has been sufficiently detailed in the body of the paper.

It may be briefly recalled that planarians experimented upon
by this method showed a certain unmistakable degree of response
which could be referable only to differences in equalized intensity.
For example, the rate of locomotion was found to be faster in any
non-directive intensity up to 431 c.m. than in darkness, although
light in itself was not always sufficient to start a worm into ac-
tivity, nor was its absence sufficient to check an animal already in
motion. Again, though no close correlation between behavior
and the degree of intensity was found to exist, there appeared cer-
tain general results which were plainly referable to intensity
differences only. Instances of such results are the behavior of
Planaria gonocephala (which was modified in several particulars
at 431 c.m. as compared with its behavior at. lower intensities) ;
the coming to rest in regions of diminished intensity of individuals
of all species except Bdelloura; and the increase of wigwag re-
sponses corresponding to an increase of intensity differences when a
field of contrasted intensities was used.

It is interesting to observe that increase in the intensity of non-
directive light, and continued exposure to non-directive light of
constant intensity, both tend to produce the same behavior that
would result in directive light. Under any of the three conditions
just mentioned there resulted by actual experiment fewer turnings,
fewer ‘indefinite changes” and more nearly straight paths on the
part of planarians than occurred when the worms were (1) placed
in non-directive light of lower intensity, (2) subjected a short time
to non-directive light of constant intensity, or (3) left in darkness.
Now, fewer turnings, fewer “indefinite changes,” and more nearly
straight paths are ordinarily characteristic results of directive
light, so that here is a case of reactions, which if resulting from the
employment of directive light would be termed phototaxis, occur-
ring in non-directive light as the result of intensity alone. Mast
(03) experimenting upon the reactions of planarians to thermal

144 Herbert Eugene Walter

stimuli obtained a similar result. He observed that apparently
“negative” as well as “positive” responses resulted when the
animals were subjected to non-directive thermal stimult.

Another noticeable phenomenon with reference to responses to

intensity is, that more wigwag responses occurred at the critical
line separating two different intensities when the lesser of the two
intensities was 16 c.m. than when it was 33 c.m. (Table XI, p. 69).
Similarly responses were more frequent when planarians were
subjected suddenly to dark than when they were flooded suddenly
by light, and, throughout a large number of series, responses were
invariably more frequent ben the worms were passing into a
region of diminished intensity than when they were entering an
area of increased intensity. It is to be inferred that all these
phases of behavior are due to the probable fact that the lower inten-
sities compared are nearer the worm’s optimum as regards light
than the higher ones, since the latter apparently have a tendency
to inhibit activity.

Lastly, the relative part played by intensity of light varies
decidedly in different species of planarians. ‘The relative inten-
sity in different parts of an aquarium, when no lens is used to les-
sen the contrast, has comparatively little influence upon Phagocata
gracilis, as its extensive wanderings (typically reproduced in
Fig. 11) toward and away from the source of light, indicate.
Planaria maculata and Planaria gonocephala, on the contrary
(Figs. 13 and 14), notwithstanding their ability to come toward the
light in the direction of the “rays” throughout the farther half of
the dish, seemed invariably to encounter an impassable barrier
as soon as they approached within a certain intensity, thereby
showing a more delicate responsiveness to intensity differences.

d The Modifying Effect of Other Factors

In attempting to analyze the relative bearing of the intensity and
of the direction of light upon the behavior of planarians there must
be constantly kept in mind two general sources of error which are
always present when these factors of light are in operation. ‘These

The Reactions of Planarians to Light 145

are (1) the physiological state of the organism at the time of obser-
vation, and( 2) the simultaneous effect of other stimuli.

A physiological state may be directly traceable to known causes,
such as previous exposure to other stimuli or the condition of meta-
bolic balance in which the animal chances to be at the time of
observation, or, again, it may be the result of factors at present
unknown, which consequently, although in active operation, are
not susceptible of analysis. In any case it is certain that the
uncontrolled factors comprehended under the term “ physio-
logical state” prove individual planarians to be not identical
Sigeha nists, but organisms possessing a more or less definite
individuality. Moreovens it has been shown that differences in
physiological state play a greater part in the determination of
behavior than do intensity differences in the light stimulus. When
a planarian is approaching a state of fatigue, for example, it
becomes indifferent to differences of intensity.

With regard to the simultaneous effect of other stimuli acting in
conjunction with light, it has already been pointed out that behav-
ior is the resultant of all the factors, external as well as internal,
which may be acting upon an organism at a given time, and that
consequently the effect of any one of the operating factors, such
as that of light, for example, cannot be determined unless the
value of the other factors involved is also taken into account.
In support of this view, which is so self-evident, it will be recalled
that some of the ways in which the responses of planarians to light
may be modified by geotaxis, thigmotaxis, goniotaxis and chem-
otaxis, were touched upon.

Summary. Direction and intensity are separable qualities of
light. Direction is dependent upon the relative positions of the
light and the organism, whereas intensity depends upon the dis-
tance between the light and the organism as well as the initial
intensity of the light.

When applied to living organisms intensity may act independ-
ently of direction, or in conjunction with it. Direction cannot
act independently of intensity upon organisms, since the latter
possess definite form and consequently cannot receive the light
at a single point.

146 Herbert Eugene Walter

With reference to an organism, directive light is resolvable
into unequalized intensity and non-directive light into equalized
intensity.

Asymmetrical intensity in directive light is largely due to the
partial shadowing of that part of the body farthest away from the
source of the light. Slight changes in the position or direction
of the light may cause considerable changes in the symmetry and
the degree of the shadow effects and consequently in the relative
intensity of the light on different regions of the body of an organ-
ism.

To different degrees of equalized or symmetrical intensity pla-
narians show considerable response, but the correlation between
their behavior and the degree of intensity is not so close as it is in
the case of asymmetrical intensity.

Increase in intensity of non-directive light, continued exposure
to non-directive light of constant intensity, and change from dark-
ness to non-directive light, all tend to bring about apparent photo-
taxis similar to that occurring in directive light.

Responses are more frequent on the part of planarians in inten-
sities approaching the optimum than in higher intensities, where
there is a tendency to inhibition. .

Relative differences in responses to various intensities are due
to specific differences between planarians.

The physiological state of an organism together with the influ-
ence of known stimuli other than light are constant sources of
error in estimating reactions to light. ‘These factors taken to-
gether play a more important part in planarian behavior than
light stimulus.

Finally, the action of light upon planarians is a function of its
intensity, which, under certain conditions, is emphasized by the
direction of the light.

2 TRIAL AND ERROR OR TROPISM?

It is apparent from the preceding section that light may have
two effects upon organisms. Of these, one is a kinetic effect,
arising from the intensity of the stimulus and resulting in a gen-

The Reactions of Planartans to Light 147

eral activity termed photokinesis, while the other, connected indi-
rectly at least with the direction from which light impinges upon an
organism, is called phototaxis. In the case of planarians these
two phases of light stimulation have been shown to be intimately
associated and both operative. Carpenter (’05) pointed out in
the case of the pomace fly that phototaxis occurs only when pre-
ceded by photokinesis or some other reaction, and such an inter-
relation of the two is undoubtedly of wide occurrence. The
object of this section is to inquire into the causes underlying pho-
totaxis. Loeb (’93b) has shown that phototaxis is the result of
orientation. It does not necessarily follow, however, that orien-
tation invariably results in phototaxis. In fact Dearborn (’oo)
found that crayfishes would orient to an electric light introduced
into the water near them without making any considerable loco-
motor movements in consequence.’

To the question of how orientation of organisms to light is
caused, three possible explanations may be presented: 1, Chance
result of photokinesis; 2, reflex response to directive stimuli;
3, voluntary action. Since the first hypothesis seems entirely
inadequate to account for the uniformity of orientation in pla-
narians, and the third alternative is out of the question with refer-
ence to these animals, a consideration of the reflex responses to
directive stimuli may be taken up at once.

There are two general theories which attempt to explain the
way in which orientation occurs through reflex responses to stimull.
‘These theories are first, the trial and error theory of Jennings and
Holmes, and secondly, the tropism theory of Verworn and Loeb.
By the trial and error theory orientation, with its consequent pho-
totaxis, 1s interpreted as the result of repeated attempts on the
part of an organism to become adjusted to any given stimulus.
Those attempts which fail to result in adjustment to the stimulus
are “errors,” and as such are followed by other attempts until
finally some one secures the necessary adjustment. ‘Trials of
this kind may be made in different ways according to the organism

7 Throughout the following discussion orientation will be understood as a position assumed with refer-
ence to the light while phototaxis will be made to include motion toward or away from the source of the
light.

148 Herbert Eugene Walter

in question. Among the infusoria and rotifera, as Jennings has
shown in a masterly series of papers,® such attempts at orientation
are made by means of a “ motor reflex,” consisting in (1) a sudden
withdrawal from the stimulus, (2) a rotation toward a structurally
defined side of the asymmetrical organism, and (3), lastly, an
advance in a new direction.

In the case of organisms which do not possess marked asym-
metry the trial and error method, as pointed out by Holmes (’o5a),
resolves itself into a series of “‘random movements;”’ that is, a
number of apparently experimental movements are made, which
finally result in the best adjustment to the stimulus.

In both of these methods the organism acts as a unit and not in
response to localized stimulation received asymmetrically.

The tropism theory, on the contrary, 1s based upon asymmetrical
action as the result of asymmetrical stimulation. If an organism
receives a stronger stimulus on one side of its body than on the
other, the result, whether direct or indirect, is that it moves in such
a way that this asymmetrical stimulation becomes symmetrical.
In other words, orientation occurs.

It is unfortunate that the tropism theory was made to apply to
the behavior of the infusoria, since it has been shown beyond
doubt by Jennings that exact observation of the behavior of these
organisms and an analysis of its details does not admit of the tropic
interpretation, but is, on the other hand, explained by the trial
and error theory of motor reflexes. It is also to be regretted that
the unquestionable rout of the tropism theory, as applied to cer-
tain protozoa and other asymmetrical forms, should have led to
an attempt to exclude it from the remainder of the animal king-
dom.

In a paper on the tropism theory Jennings (’o4a) names as an
essential criterion of tropism the direct unilateral stimulation of
the motor organs. After showing how inadequate such an assump-
tion is to explain the orientation of animals, particularly that of
Infusoria, he continues (’o4a, p. 104), “We should perhaps con-

® See bibliography in Contributions to the Study of the Behavior of Lower Organisms. Carnegie
Inst. of Washington. Publication No. 16. 256 pp. 1904.

The Reactions of Planarians to Light 149

sider here a modification of the original form of the tropism theory
that has been proposed by some authors. ‘This is in regard to
the assumption that the stimulating agent acts directly on the
motor organs upon which it impinges. For this it is sometimes
proposed to substitute the view that the action of the stimulating
agent is directly on the sense organs of the side on which the stim-
ulus impinges and only indirectly on the motor organs through
their nervous connection with the sense organs. When thus modi-
fied the theory of course loses its simplicity and its direct explain-
ing power, which made it so attractive. In order to retain any of
its value for explaining the movements of organisms, it would
have to hold at least that the connections between the sense organs
and the motor organs are of a perfectly definite character so that
when a certain sense organ is stimulated a certain motor organ
moves in a certain way. When we find, as we do in the flatworm
(see the following paper), that to the same stimulus on the same
part of the body, under the same external conditions the animal
reacts sometimes in one way, sometimes in another, the tropism
theory, of course, fails to supply a determining factor for the
behavior.”

It seems to me that the mechanism by means of which the
asymmetrical response is brought about is immaterial, so long as
that response can be shown to be the result of asymmetrical stimu-
lation. Asymmetrical response might occur either from direct
stimulation of the motor organs as was implied in the earlier
papers on the infusoria, or by means of a more complex method,
consisting of stimulation of the sense organ, transmission to the
central nervous system and thence to the motor organs.

The outcome in either case would fulfill the demands of the trop-
ism theory, if asymmetrical response to asymmetrical stimulation
be taken as its criterion. In the quotation just cited, the objection
that such transmission compels stereotyped behavior is hardly
valid, since stereotyped reaction is by no means the only alterna-
tive of asymmetrical stimulation. ‘That flatworms do not respond
uniformly to directive stimuli cannot be disputed, but that fact
does not exclude the possibility of all tropic reaction on their part.
The imperfection of response may be simply the result of imper-

150 Herbert Eugene Walter

fections in the worm’s nervous circuit, assuming that planarian
reactions are due to indirect rather than to direct stimulation of
the motor organs. In fact, repeated evidence of the failure of a
constant and perfectly invariable orientation on the part of pla-
narians has been given in the preceding pages. Such failure,
moreover, is quite as likely to occur in the application of the trop-
ism theory to behavior as it is in the case of the trial and error
theory, since stereotyped reactions and forced movements, as
Holmes (05a, p. 112) has emphasized, are no more characteristic
of tropisms, which depend upon a differentiated stimulation and
response, than they are of trial and error movements, resulting
from a single motor reflex given in response to all kinds of stimu-
lation.

Furthermore, it has been urged that tropism indicates a simpler
form of reaction than trial and error for the reason that it in-
volves only a local part of an organism while the motor reflex of
trial and error requires that the organism act asa whole. Conse-
quently, since motor reflex has been indisputably demonstrated
as the method of infusorian phototaxis, Jennings (’04a, p. 95) asks,
“Should we conclude that the reactions in the higher metazoa are
simpler and less unified than in the protozoa f”

That the motor reflex, which occurs with machine-like uni-
formity, regardless of the point where the stimulation is received,
is more complex in character than the stimulation of an asym-
metrical part of an organism which may depend for its response
upon sense-organ, nervous transmission and motor apparatus
is an assumption difficult to sustain. It seems more reasonable
to agree with Harper (05) in placing tropism higher in the evolu-
tionary scale than trial and error.

The fallacy that “tropism leads nowhere; it is a fixed final thing
like a crystal’ (Jennings, ’o4c, p. 251), while trial and error alone
offers possibilities of the higher evolution of phototaxis, has already
been answered by Holmes, who points out that trial and error,
at least that phase of trial and error depending upon motor reflex,
is even more fixed and stereotyped than the reactions occurring
in accordance with the tropism schema. To quote: “The end
result of both methods is the same, 7. ¢., to get the organism away

The Reactions of Planarians to Light 151

from the stimulus. In the one case it is accomplished by direct
reflex without more ado; in the other, only after a considerable
waste of energy in inconsequential vermiculations” (Holmes,
"05a, p- 110).

It is at least conceivable that under the tropism schema, as the
nervous differentiation of an animal becomes more complete, the
ability of the organism to interfere with and modify its machine-
like responses to external stimuli might also increase, resulting
in a flexibility of behavior which would present quite as much
variation for natural selection to act upon as that evolved by the
trial and error method. ‘This point of view by no means denies
that trial and error is the usual “method of intelligence” (C. L.
Morgan ’o0, p. 139). It is simply an attempt to recognize in the
method of tropism also one of the possibilities of evolutionary
progress in behavior and as such holding a higher position in the
scale of evolutionary methods than trial and error by motor reflexes,

It has been shown (p. 143) that planarian responses of an appar-
ently asymmetrical character may occur as a result of symmetri-
cal stimulation. Similar instances in the case of planarians have
also been demonstrated by Mast (’03) with reference to thermal
stimuli. This, however, is no exception to the validity of the tro-
pism theory, in which asymmetrical responses result from asym-
metrical stimulation. Because a planarian may make an appar-
ently phototropic response when subjected to symmetrical stim-
ulation, is not evidence against the supposition that the usual
phototropic response is due to asymmetrical stimulation.

The “wigwag’’ movements of planarians, to which repeated
reference has been made in the preceding pages, resemble super-
ficially the “random movements” of the earthworm as described
by Holmes. They do not, however, ordinarily appear to be the
basis of trial and error selection resulting in orientation, since
in a majority of cases, after a worm Hales and makes wigwag
movements it continues on its way without a change of direction.
The movements of Bdelloura candida, as shown in Fig. 12
form an exception to ordinary planarian behavior in this respect

As a rule wigwag movements are probably occasioned by a
general Eeambence arising from some stimulation which thowrs

152 Herbert Eugene Walter

the worm into a different physiological state. Exploring move-
ments, such as these seem to be, may bring about asymmetrical
stimulation, in which case the worm makes a tropic response.

It was particularly noticed that when planarians received light
from below, the anterior end of the body was frequently tilted
back and forth as if to make it possible for the light when coming
from such an unusual direction to enter the pigment cups of the
eyes. The phenomenon suggested the craning of necks and bob-
bing of heads among a crowd of people who are all trying to see
the same object at once.

Wigwag movements seem to be oftener connected with changes
in the intensity of light than with changes in its direction. When
the latter occur, tropic response is immediately the result.

In the course of the experiments previously described wherein
the worms glided from an area of one intensity of non-directive
light into another it was noticed that in a majority of cases when
the critical line was not crossed at right angles, no change in course
occurred, even when the worm halted and made wigwag move-
ments. Of course at a certain instant of any diagonal crossing
of the critical line one eye must receive more stimulus than the
other, in which case according to an inflexible tropism theory
asymmetrical response ought to occur. But such a response does
not frequently appear aa the reason for this becomes clear when
it is remembered that a considerable number of responses were
shown to occur which were called “latent wigwags”’ (Fig. 4, £), be-
cause they failed to make their appearance until in some instances
the worm had passed more than the length of its body beyond
the critical line. Since, therefore, latency of response to intensity
is by no means uncommon, it is evident that the brief interval of
asymmetrical stimulation occurring when a worm glides diagonally
into an area of different intensity is not sufficient to result in an
asymmetrical response.

Two conclusions, then, seem reasonable, namely, that phototaxis
as related to planarians is primarily due to asymmetrical response
resulting from asymmetrical stimulation, and that wigwag move-
ments, together with similar apparent trial and error forms of
behavior, contribute chiefly to this end, 7. ¢., to phototaxis.

The Reactions of Planarians to Light 153

Summary. Orientation may occur without phototaxis.

Two theories have been advanced to explain orientation and
phototaxis in lower organisms, namely, the trial and error theory
and that of the tropisms. “The former may be based upon “‘ motor
reflexes” or upon “random movements” according to the sym-
metry of the animal.

The tropism theory rests upon asymmetrical response to asym-
metrical stimulation. It does not necessarily depend upon the
direct stimulation of the motor organs, nor is it essentially stereo-
typed in its character any more than are trial and error responses
by motor reflex or random movements.

The tropic form of response may, and probably does, require a
more complex mechanism than that which causes the motor reflex,
consequently it is the form of response to be logically expected
among planarians, since the motor reflex has been proven to be
the form utilized by the protozoa.

Tropisms, as well as trial and error movements, provide, through
the modifying control of an evolving central nervous system, sufh-
cient latitude of variation for natural selection to work upon in the
evolution of higher forms of behavior.

Asymmetrical response may, in certain cases, result from sym-
metrical stimulation, but ordinarily its cause is asymmetrical
stimulation.

Wigwag movements are occasioned most frequently by changes
in intensity, and they may result in orientation and phototaxis
by assisting an organism to secure asymmetrical stimulation.

Latency of reaction accounts for some of the failures in orien-
tation which often occur even when asymmetrical stimulation is
acting upon an organism.

Finally, the orientation and phototaxis of planarians is more
consistently explained by the theory of tropisms than by the theory
of trial and error.

3. ADAPTATION.

It remains, finally, to inquire how far the reactions of planarians
to light are adaptive; that is, how far the response to light is “of

154 Herbert Eugene Walter

such a kind that it better insures the existence of the individual,
or of the race” (T. H. Morgan ’o03, p. 1).

It is evident that the generally negative character of the reac-
tions of planarians to light indicates a tendency on the part of
these worms to reduce as much as possible the amount of light
stimulation received or to avoid it altogether. The rigor effects
of excessive stimulation furnish evidence also that light is a factor
in a planarian’s environment which it finds unavoidable and
unwelcome and to which it is adapted only in a negative fashion.
In fact the vague distinction separating “lower” from “higher”
animals consists largely in the ability of higher animals to assume
an active aggressive rather than a passive defensive relation toward
the factors making up their environment. For example, the
evolution in animals of the visual organs, which in the planarians
is only inceptive, enlarges the possible range of photic responses
until light becomes an essential factor in an animal’s environment,
contributing largely to its welfare by enabling it to see its food, to
avoid its enemies and to select its mates. It is plain that light
plays no such important part in the activities of planarians, for,
as has already been pointed out, light per se is not essential to pla-
narians, since they are known to ihe successfully in dark caves.
Moreover, so far as known, light does not influence the regenera-
tive or reproductive processes of planarians in any way whatso-
ever. The formation of pigment may perhaps be regarded as an
adaptation to light conditions, inasmuch as anna possessing
pigment are thereby shielded to a certain degree from excessive
stimulation.

With reference to activities connected with nutrition and repro-
duction, planarians are not dependent upon light stimulation.
They are otherwise equipped, since they doubtless find their food
by chemotactic means and avoid whatever enemies they may have,
not aggressiv ely nor activ ely by retreating from visible foes but malen
in a passive way by remaining Concealed from enemies that might
see them. They have no organs of defense but survive by escap-
ing attention. In this sense their negative phototaxis may be
regarded as of protective value and consequently adaptive.

Furthermore, the geographical distribution of fresh water pla-

The Reactions of Planarians to Light 155

narians has been shown by Borelli (’93) and Wilhelmi (’04) to be
chiefly dependent upon temperature and almost not at all upon the
amount of illumination to which they are subjected. Voigt (’04)
noticed that worms when hungry may be seen wandering about
even in patches of bright sunlight with apparent disregard of light.
This seems to be a case of the light reactions becoming over-
balanced by other responses.

Summary. Light is not an essential factor’ in planarian
activities, since the behavior necessary to the welfare of the
individual and the race is mainly referable to other factors.

A planarian’s response to light is of a passive character, which
may have an adaptive significance only in so far as its phototaxis
tends to conceal the worm from its enemies. The presence of
pigment may also be regarded as an adaptive condition induced
by the animal’s relation to light.

The evolution of the photoreceptive apparatus of the planarian
has not reached the degree of differentiation necessary to enable it
to secure for itself such adaptations to the factor of light in its
environment as would make aggressive activity possible to it in a
manner characteristic of higher animals.

VII BripLlioGRAPHY

ARLOING, S., ’87a—| Letter on Mechanism of Destruction of Microbes by Light. |
Ann. Inst. Pasteur., vol. i, pp. 594-596.
*87b—Les spores du Bacillus anthracis sont réellement tuées par la
lumiére solaire. Comp. Rend. Acad. Sci., Paris, tom. 104, pp.
701-703.
BarDEEN, C. R., ’ora—On the Physiology of the Planaria maculata with especial
Reference to the Phenomena of Regeneration. Amer. Jour.
Physiol., vol. v, no. 1, pp. 1-55.
‘otrb—The Function of the Brain in Planaria maculata. Amer. Jour.
Physiol., vol. v, no. 3, pp. 175-179.
BartHELeémy, A., °84—Sur la physiologie d’une Planaire verte (Convoluta
Schultzii). Comp. Rend. Acad. Sci., Paris,tom. 99, pp- 197-200.
Beer, T., ’01—Ueber primitive Sehorgane. Wiener klin. Wochenschr., 1gor,
Nr. 11-13.

156 Herbert Eugene Walter

Bercer, F. W., ’oo—Physiology and Histology of the Cubomeduse. Mem.
Biol. Lab., Johns Hopkins Univ., vol. iv, pt. 4, pp. 1-84, pl. 1-3.
Birce, E. A., ’97—The Vertical Distribution of the Limnetic Crustacea of Lake
Mendota. Biol. Centralbl., Bd. 17, Nr. 10, pp. 371-374.
Boun, G., ’03a—Sur les mouvements oscillatoires des Convoluta roscoffensis.
Comp. Rend. Acad. Sci., Paris, tom. 137, pp. 576-578.
’o3b—Sur le phototropisme des Artiozoaires supérieurs. Comp. Rend.
Acad. Sci., Paris, tom. 137, pp. 1292-1294.
’03c—Les Convoluta roscoffensis et la théorie des causes actuelles.
Bull. Mus. Hist. Nat., Paris., tom. 9., pp. 352-364.
Bore ut, A., ’93—Osservazioni sulla Planaria alpina (Dana) e catalogo dei Den-
droceli d’aqua dolce trovati nell’ Italia del Nord. Boll. Mus.
Zool. ed Anat. comp. R. Univ. Torino, vol. viii, no. 137, pp.
T—03.
Bucuner, H., ’92—Ueber den Einfluss des Lichtes auf Bakterien. II. Mittheilung.
Centralbl. f. Bakteriol. u. Parasitenk., Bd. 12, No. 7, pp. 217-219.
CarpENTER, F. W.,’05—The Reactions of the Pomace Fly (Drosophila ampelophila
Loew) to Light, Gravity and Mechanical Stimulation. Amer.
Nat., vol. xxxix, no. 459, pp. I157—-I7I.
CarriERE, J., ’82—Die Augen von Planaria polychroa Schmidt und Polycelis
nigra Ehrb. Arch. f. mikr. Anat., Bd. 20, pp. 160-174, Taf. 9.
CurcukorF, G. D.,’92—Recherches sur les Dendrocoeles d’eau douce (Triclades).
Arch. de Biol., tom. 12, pp. 435-560, pl. 15-20.
Coun, F., ’53—Ueber eine Gattung aus der Familie der Volvocinen. Zeit. f.
wiss. Zool., Bd. 4, pp. 77-116.
’64—Ueber die Gesetze der Bewegung mikroskopischer Thiere und
Pflanzen unter Einfluss des Lichtes. Jahresber. Schles. Gesell-
sch. f. vaterl. Cultur, Bd. 42, pp. 35-36.
Cottiyn, A., ’91—Mittheilung uber Planaria alpina (Dana). Sitzungsber.
Gesellsch. naturf. Freunde, Berlin, Jahrg. 1891, Nr. 9, pp.
177-180.
Curtis, W. C., ’°02—Life History and Reproduction of Planaria maculata.
: Proc. Boston Soc. Nat. Hist., vol. xxv, pp. 515-559.
DatyeELL, J. C., ’14—Observations on Some Interesting Phenomena in Animal
Physiology, Exhibited by Several Species of Planaria, illustrated
by colored figures of living animals. Edinburgh, 8°, xi + 151
Pp:s tpl.
’*53—The Powers of the Creator Displayed inthe Creation; or, Observa-
tions on Life Amidst the Various Forms of the Humbler Tribes
of Animated Nature. London, 4°, vol. 11, 359 pp., 46 pl.

The Reactions of Planarians to Light 157

DanpriEu (DE Carcassonne), ’88—Influence de la Lumiére dans la Destructions
des Bactéries; pour servir a Etude du “Tout a l’Egout.” Ann.
d’Hyg. pub. et de Méd. lég., Paris, sér. 3, tom. 20, no. 5, pp.
448-451.

Darwin, C., ’44—Brief Descriptionsof Several Terrestrial Planariz and of Some
Marine Species, with an Account of their Habits. Ann. and Mag.
Nat. Hist., vol. xiv, pp. 241-251, 1 pl.

*81—The Formation of Vegetable Mold through the Action of Worms,
with Observations on their Habits. New York, 12°, 326 pp.

Davenport, C. B., anp Cannon, W. B., ’97—On the Determination of the
Direction and Rate of Movement of Organisms by Light. Jour.
of Physiol., vol. xxi, no. I, pp. 22-32.

Dearsorn, G. V. N., ’o0o—Notes on the Individual Psycho-physiology of the
Crayfish. Amer. Jour. Physiol., vol. iii, no. 9, pp. 404-433.

Dexace, Y., ’86—Etudes histologique sur les Planaires rhabdocceles acceles
(Convoluta Schultzii O.Schm.). Arch. Zool. Expér. et Gén., sér.
2, vol. iv, pp. 109-144, pl. 5-6.

Downes, A., AND Brunt, T. P., ’77—Researches on the Effect of Light upon
Bacteria and other Organisms. Proc. Roy. Soc., London, vol.
xxvi, pp. 488-500.

*78—On the Influence of Light upon Protoplasm. Proc. Roy. Soc.,
London, vol. xxvii, pp. 199-212.
Ducraux, E., ’85a—Influence de la lumiére du soleil sur la vitalité des germes de
microbes. Comp. Rend. Acad. Sci., Paris, tom. 100, pp. 119-121.
’85b—Influence de la lumiére du soleil sur vitalité des micrococcus.
Comp. Rend. Acad. Sci., Paris, tom. 101, pp. 395-3098.
*90—Action de la Lumiere sur les Microbes. ‘ Revue critique. Ann. Inst,
Pasteur, vol. iv, pp. 792-800.

Duces, A., ’28—Recherches sur l’organization et les moeurs des Planariées.
Ann. Sci. Nat., tom. 15, pp. 139-183.

Enceitmann, T. W., ’79—Ueber Reizung contraktilen Protoplasmas durch
plotzliche Beleuchtung. Arch. f. ges. Physiol., Bd. 19, pp. 1-7.

*83—Bacterium photometricum. Ein Beitrag zur vergleichenden Physi-
ologie des Licht-und Farbensinnes. Arch. f. ges. Physiol., Bd.
30, pp. 95-124, Taf. 1.

Famintzin, A., ’67—Die Wirkung des Lichtes auf Algen und einige andere ihnen
nahe verwandte Organismen. Jahrb. f. wiss. Bot. (Pringsheim),
Bd. 6, pp. 1-48, Taf. 1-3. ;

FranpsEn, P., ’01—Studies on the Reaction of Limax maximus to Directive Stim-
uli. Proc. Amer. Acad. Arts and Sci., vol. xxxvii, pp. 185-227.

158 Herbert Eugene Walter

Fiuner, N., ’o6—Notizen zur Biologie von Convoluta roscoffensis Graff. Biol.
Centralbl., Bd. 26, Nr. 1, pp. 24-26.

GamB LE, F. M., anp KEEBLE, F., ’03—The Bionomics of Convoluta roscoffensis,
with Special Reference to its Green Cells. Quart. Jour. Micr.
Sci., vol. Ixvii, pp. 363-431, pl. 30-31.

Geppes, P., ’79—Observations on the Physiology and Histology of Convoluta
Schultzii. Proc. Roy. Soc., London, vol. xxvill, pp. 449-457-

Giss_Ler, C. F., ’°82—A Marine Planarian and its Habitation. Amer. Nat., vol-
XVI, 00: Wy pp: §2=53;, gs 1-2.

Graber, V., °83—Fundamentalversuche iiber die Helligkeits- und Farbenemp-
findlichkeit augenloser und geblendeter Thiere. Sitzungsb. d.
Wien, Akad., Bd. 87, pp. 201-236.

GrarF, L. von, ’84—Zur Kenntnis der physiologischen Function des Chloro-
phylls im Thierreich. Zool. Anz., Bd. 6, Nr. 177, pp. 520-527-

Groom, T. T., unp Logs, J.,’g0—Der Heliotropismus der Nauplien von Balanus
perforatus und die periodischen Tiefenwanderungen pelagischer
Tiere. Biol. Centralbl., Bd. 10, Nr. 5, pp. 160-177.

Haper.anpt, G., ’91—Ueber den Bau und die Bedentung der Chlorophyllzellen
von Convoluta roscoffensis. Appendix (pp. 75-90) to L. von Graff,
Die Organization der Turbellaria accela. Leipzig. 4°, Holzsch.
u. go Taf.

Harper, E. H., ’05—Reactions to Light and Mechanical Stimuli in the Earth-
worm Pericheta bermudensis (Beddard). Biol. Bull., vol. x,
no. 1, pp. 17-34.

Hesse, R., ’96—Untersuchungen iiber die Organe der Lichtempfindung bei
niederen Thieren. I. Die Organe der Lichtempfindung bei Lum-
briciden. Zeit. f. wiss. Zool., Bd. 61, pp. 393-419, Taf. 20, 1 Fig.

97—Untersuchungen iiber die Organe der Lichtempfindung bei
niederen Thieren. I]. Die Augen der Plathelminthen, insonder-
heit der tricladen Turbellarien. Zeit. f. wiss. Zool., Bd. 62, pp-
527-582, Taf. 27 u. 28, 3 Fig.

Hopeg, C. F., anp A1kens, H. A.,’95—The Daily Life of a Protozoan: A Study
in Comparative Psycho-physiology. Amer. Jour. Psychol., vol.
vi, no. 4, pp. 524-533.

Hoae, C. ’97—On Bipalium kewense, Moseley. Trans. Nat. Hist. Soc., Glasgow,
n. s., vol. v, pt. I, pp. 53-54.

Houmes, S. J. ’o1—Phototaxis in the Amphibia. Amer. Jour. Physiol., vol. v,
no. 4, pp. 211-234.

’0o3—Phototaxis in Volvox. Biol. Bull., vol. iv, pp. 319-326.

The Reactions of Planarians to Light 159

Homes, S. J., ’05a—The Selection of Random Movements as a Factor in Photo-
taxis. Jour. Comp. Neurol. and Psychol., vol. xv, no. 2, pp.
98-112.
’o5b—The Reactions of Ranatra to Light. Jour. Comp. Neurol. and
Psychol., vol. xv, pp. 305-349-
Hott, E. B., and Ler, F. S., ’01—The Theory of Phototactic Response. Amer.
Jour. Physiol., vol. iv, no. 9, pp- 460-481.
Irma, I., ’84—Untersuchungen tiber den Bau und die Entwickelungsgeschichte
der siisswasser Dendrocoelen (Tricladen). Zeit. f. wiss. Zool.,
Bd. 40, pp. 359-464, Taf. 20-23.
JAnincuin, E., ’96—Beitrage zur Kenntnis des Turbellarien Auges. Zeit. f.
wiss. Zool., Bd. 62, pp. 250-288, Taf. 10-11, 7 Texthg.
Jamieson, J., 82—The Influence of Light on the Development of Bacteria.
Nature, vol. xxvi, pp. 244-245.
Janowsk1, T., ’90—Zur Biologie der Typhusbacillen. Die Wirkung des Sonnen-
lichts. Centralbl. f. Bakt. u. Parasitenk., Bd. 8, Nr. 6, pp. 167-
172; Nr. 7, 193-199; Nr. 8, 230-234; Nr. 9, 262-266.
Jennies, H. S.,’04a—The Theory of Tropisms. Carnegie Inst. of Washington,
Pub. no. 16, pp. 89-107.
’o4b—Physiological States as Determining Factors in the Behavior of
Lower Organisms. Carnegie Inst. of Washington, Pub. no. 16,
pp- 109-128.
’o4c—The Method of Trial and Error in the Behavior of Lower Organ-
isms. Carnegie Inst. of Washington, Pub. no. 16, pp. 237-252.
KennEL, J. von, ’88—Untersuchungen an neuen Turbellarien. Zool. Jahrb.
Abt. f. Anat. und Ont., Bd. 3, pp. 447-486, Taf. 18-19.
Lana, A., ’84—Die Polycladen (Seeplanarien) des Golfes von Neapel und der
angrenzende Meeresabschnitte. Fauna u. Flora d. Golfes yon
Neapel. XI. Monographie. x+688 pp. 39 Taf. Leipzig.
Lennert, G. H., ’91—Beobachtungen an Landplanarien. Arch. f. Naturg.,
Jahrg. 57, pp- 306-350.
Lemy, J.,’58—Note on Finding Rhincodemus sylvaticus at Night. Proc. Acad.
Nat. Sci., Philadelphia, 1858, pp. 171-172.
Linu, F. R., ’°o1r—Notes on Regeneration and Regulation in Planarians. Amer.
Jour. Physiol., vol. vi, no. 2, pp. 129-141.
Logs, J., ’90—Der Heliotropismus der Thiere und seine Uebereinstimmung mit
dem Heliotropismus der Pflanzen. Wirzburg, 8°, 118 pp.
’93a—On the Influence of Light on the Periodical Depth-migrations of

Pelagic Animals. Bull. U. S. Fish Com., vol. xiii, pp. 65-68.

160 Herbert Eugene Walter

Logs, J., ’93b—Ueber kiinstliche Umwandlung positiv heliotropischer Thiere
in negativ heliotropische und umgekehrt. Arch. f. ges. Physiol.,
Bd. 54, pp. 81-107, 6 Holzsch.
*94—Beitrage zur Gehirnphysiologie der Wiirmer. Arch. f. ges. Physiol.,
Bd. 56, pp. 247-269, Fig. 1-4.
Mast, S. O., ’03—Reactions to Temperature Changes in Spirillum, Hydra and
Fresh-Water Planarians. Amer. Jour. Physiol., vol. x, no. 4,
pp- 165-190.
Maupas, E., ’87—Sur la puissance de multiplication des Infusories ciliés. Comp.
Rend. Acad. Sci., Paris, tom. 104, pp. 1006-1008.
Morean, C. L., ’0o—Animal Behavior. London, 8°, 344 pp.

Morean, T. H., ’03—Evolution and Adaptation. New York, 8°, x+470 pp-

Mose ey, H. N., ’74—On the Anatomy and Histology of the Land Planarians
of Ceylon; with Some Account of their Habits, and a Description
of Two New Species, and with Notes on the Anatomy of Some
European Aquatic Species. Phil. Trans. Roy. Soc. London, vol.
clxiv, pp. 105-171, pl. 10-15.

NaceL, W. A., ’94—Beobachtungen iiber den Lichtsinn augenloser Muscheln.
Biol. Centralbl., Bd. 14, Nr. 11, pp. 385-390.

Ortmanns, F., ’92—Ueber die photometrischen Bewegungen der Pflanzen.
Flora, Bd. 75, pp. 183-266, Taf. 4.

PansInI, ’89—Action de la Lumiere solaire sur les micro-organismes. Ann. Inst.
Pasteur, tom. 3, pp. 686.

Parker, G. H., ’02—The Reactions of Copepods to various Stimuli, and the
Bearing of this on Daily Depth Migrations. Bull. U.S. FishCom.,
IgOI, pp. 103-123.

’03—The Phototropism of the Mourning-cloak Butterfly, Vanessa

antiopa Linn. Mark Anniversary Vol., pp. 453-469, pl. 33-

Parker, G. H., anp Arkin, L., ’or—The Directive Influence of Light on the
Earthworm Allolobophora fcetida Sav. Amer. Jour Physiol., vol.
Vv, NO. 3, pp. 151-157

Parker, G. H., anp Burnett, F. L., ’00o—The Reactions of Planarians, with
and without Eyes, to Light. Amer. Jour. Physiol., vol. iv, no. 8,
Pp. 373-385, fig. 1-4.

Peart, R., ’03—The Movements and Reactions of Fresh-water Planarians: A
Study of Animal Behavior. Quart. Jour. Micr. Sci., vol. xlvi, pp.
50957 74:

Peart, R., and Cote, L. J.,’02—The Effect of very Intense Light on Organisms.

Michigan Acad. Sci., Third Report, pp. 77-78.

The Reactions of Planartans to Light 161

Rant, E., ’01—Ueber den Phototropismus einiger Arthropoden. Biol. Centralbl.,
Bd. 21, Nr. 3, pp. 75-86.

Raum, J., °89—Der gegenwartige Stand unserer Kenntnisse iiber den Einfluss des
Lichtes auf Bakterien und auf den thierischen Organismus. Zeit.
f. Hyg., Bd. 6, pp. 312-368.

Roux, E., ’87—De l’action de la lumieére et de |’air sur les spores de la bactéridie
du charbon. Ann. Inst. Pasteur, tom. I, pp. 445-452.

ScHouTEDEN, H., ’02—Le Phototropisme de Daphnia magna Straus (Crust.).
Ann. Soc. ent. Belgique., tom. 46, pp. 352-362.

Scuuttze, M., ’51—Beitrage zur Naturgeschichte der Turbellarien. Erste Abt.
Greifswald, 4°, 78 pp. 7 Taf.

SeKeERA, E., ’03—Einige Beitrage zur Lebensweise von Vortex helluo (viridis M.
Sch.) Zool. Anz., Bd. xxvi, no. 708, pp. 703-710.

Situ, A. C., ’02—The Influence of Temperature, Odors, Light and Contact on
the Movements of the Earthworm. Amer. Jour. Physiol., vol.
vi, no. 7, pp. 459-486.

StrasBurGeRr, E., ’78—Wirkung des Lichtes und der Warme auf Schwarmsporen.
Jena. Zeit., Bd. 12, pp. 551-625.

Tow te, E. W., ’0o—A Study in the Heliotropism of Cypridopsis. Amer. Jour.
Physiol., vol. ili, no. 8, pp. 345-365.

Tynva tL, J., ’778—Note on the Influence Exercised by Light on Organic Infusions.
Proc. Roy. Soc., London, vol. xxviii, pp. 212-213.

Verworn, M., ’o1—Allgemeine Physiologie. 2te Aufl. Jena, 8°, 631 pp.

Voict, W., ’04—Ueber die Wanderungen der Strudelwiirmer in unseren Gebirgs-
bachen. Verh. naturh. Vereins preuss. Rheinlande, Westfalens
und Regierungsbez. Osnabriick, Jahrg. 61, pp. 102-178.

Watter, H. E., ’06—The Behavior of the Pond Snail; Lymnzus elodes Say-
Brooklyn Inst. Arts and Sci., Cold Spring Harbor Monographs,
VI, 35 pp-

Warp, H. M., ’94—Further Experiments on the Action of Light on Bacillus
anthracis and on the Bacteria of the Thames. Proc. Roy. Soc.,
London, vol. lvi, pp. 315-374.

Wuirman, C. O., ’99—Animal Behavior. Wood’s Hole Lectures for 1898.
Boston, pp. 285-338.

WitueEmt, J., °o4—Beitrage zur Kenntnis der Verbreitung und Biologie der
Siisswassertricladen. Zool. Anz., Bd. xxvii, Nr. 11, pp. 355-365;
Nr. 12, 369-375.

Wi.eM, V.,’91—La vision chez les gasteropodes pulmonés. Comp. Rend. Acad.
Sci., Paris, tom. 112, pp. 247-248.

162 Herbert Eugene Walter

Woopwortn, W. McM., ’97—Contributions to the Morphology of the Turbel-
laria: I]. On some Turbellaria from Illinois. Bull. Mus. Comp.
Zool. Harvard Coll., vol. xxxi, pp. 1-16, pl. 1.

Yerkes, R. M., ’99—Reaction of Entomostraca to Stimulation by Light. Amer.
Jour. Physiol., vol. iii, no. 4, pp. 157-182.

’03.—A Study of the Reactions and Reaction Time of the Medusa

Gonionema Murbachii to Photic Stimuli. Amer. Jour. Physiol.,
vol. 1x, no. 5, pp. 279-307.

ZELENY, C., '05—The Rearing of Serpulid Larva with Notes on the Behavior of
the Young Animals. Biol. Bull., vol. viii, no. 5, p. 308.

REGENERATION IN COMPOUND EYES OF
CRUSTACEA

MARY ISABELLE STEELE, M.A.

Witu Sixteen Pirates AND Two Ficures IN THE TExT

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IV The preliminary regenerative processes...... 26200. 2. 0 cee sence ese ete ese c teers eee ees 172
A Healing of the wound and formation of new cuticle.............-.--.0.00-02.0-055 173
Bae Removaljotthenny ured tissue cjasiajis rete aie\a/aicisvace <ratesares ses aiata atagstolecovafafcvatcrmiagaate/ starerayere 179
CmiMethodloficellidiysistomt terete retcts ates sectors sever sieves sheieteleye ater relia aiciatelsiexstateve/seievatescta (ao 183
V_ Regeneration occurring after destruction of upper part of eye ........- 0-000. eee eee eee 185
Ac Repenerationlofifinctional eyez ioi.)..c/- o)o/~\+ 101 55+ (0/2 e1e\-1210.vipie nhs e}sinseiels ee [esereieiciessisisiotsiaie/ aie 185
meme epeneratinpieye trom Suita ce PLOW ate tere alm ayate lore <1 2.51= beta ials\cletet-i-.s erepasiieioresetete 186
2 Details of the development of regenerating ommatidia.....................-. 190
a) regeneration ofiretinulee = <2. <eteeine1s ec sie ences = Seed cron Sats tee ee 190
6) WRegeneration‘oficrystalline cOneS s<.s\e:ci ersie «ccs misug vicieie /aicieiels a. tieyainte/aiete 192
c Regeneration of rhabdoms
3 Comparison of ommatidia in regeneration and ontogeny...........-.--.-..-.-
4 Differences in the regeneration of eyes among Palemonetes Crangon and
Grn Gard Sxpppba0 550 odgnd0 odup so bodosuocdennsccptudasonsudoa 199
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B Cases of aberrant regeneration of ommatidia .......5.2+.c0serececscecsercercecses 203
C_ Eye stumps that show an abnormal development or no regeneration................- 210
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2 Eye stumps that show no regeneration.......-.----+--++.s+5+ APopabteean 2s 207)
VI Regeneration after removal of the yreater part or all of the optic ganglion................ 219
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2 Cases that show no special regeneration...........+-6+2+-ce erect sees ences 221
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E General consideration of the regeneration following removal of entire eye........-..-- 230
VIE Regeneration after splitting the eye longitudinally......... 26.60.0600 eee e eee eee eee eee 237
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TDG EET B00 6d denned dup pokdesueuUDABCOeesnaaocur cabsemapgnoeEb concen Sanmodeon 241
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Tue Journat or ExreriMENTAL ZOOLOGY, VOL. V, NO. 2.

164 Mary Isabelle Steele

I INTRODUCTION

The regeneration of the Crustacean compound eye has been
made a subject of observation by a number of investigators. But
for the most part end results alone have been described. Very
few details of the processes involved in regeneration have been
given. ‘To give an accurate description of the histogenesis of the
regenerated structures is the chief aim of this paper in the belief
that it will contribute something toward a more accurate under-
standing of the more general problem of regeneration.

The problem has separated itself into three main divisions:
first, the regeneration of a functional eye; second, a search for the
causes of no regeneration and abnormal regeneration, and third,
observations upon the heteromorphic regeneration which may
follow the removal of the entire eye.

Herbst (’96, 00) and Morgan (’98) have made the principal
observations upon the regeneration of the Crustacean compound
eye. But so far as the particular phases dealt with in this paper
are concerned Herbst’s descriptions are not sufficiently detailed
to be of especial assistance in this work. Herbst’s observations
upon the regenerated heteromorphic structures are somewhat
more extensive and in some respects furnish an excellent basis for
comparison with the results to be discussed in this paper. For
the most part, however, where results have been compared with
the work of others the comparison is made between the regenera-
tive and embryonic development and between normal and regen-
erated structures.

The terminology employed is in great part that used by Parker
(91) in his work upon the compound eyes of Crustacea, it being
the terminology in most general use. A few minor deviations
have been made but no wholly new terms have been introduced.

The following plan has been followed in this paper. First, a
brief description of the normal adult eye is given to furnish a basis
for comparison. ‘This is followed by a description of the prepara-
tory regenerative processes which in reality constitutes one of the
most important phases of the subject. Then the regenerative
processes proper are described under the three main divisions
already suggested.

Regeneration in Compound Eyes of Crustacea 165

The experimental part of the work was begun at the University
of Pennsylvania. Observations upon living material were also
carried on during two summers at Woods Hole, Mass., and one
summer at Cold Spring Harbor, L. I. The greater part of the
detail work of examining the preserved material has been done at
the University of Missouri during the present year.

In closing I wish to express my thanks to Dr. E. F. Phillips and
Dr. D. B. Casteel for the care of experiments; to Dr. E. G. Conklin,
University of Pennsylvania; Dr. C. B. Davenport, Carnegie
Institute at Cold Spring Harbor, and Dr. George Lefevre and
Dr. W. G. Curtis, of the University of Missouri, for their interest
and valuable suggestions during the course of the work.

II Marerrat anp Metuops

The small hermit crab, Eupagurus longicarpus, the common
shrimp, Palzmonetes vulgaris, and the sand shrimp, Crangon
vulgaris, afford the greater part of the material used in the series
of experiments to be described in this paper. For comparison
two species of crayfish, Cambarus virilis and C. gracilis, a species
of fresh water Ascellus, the common (wood-louse), Oniscus, and
the fresh water Gammarus were used. Other emacs also
were experimented upon but since no decisive results were obtained
they need not be considered here.

The work has been confined chiefly to the eyes of the forms
used although experiments upon the appendages, particularly the .
antennz, were conducted at thesame time. “These were, however,
largely for the purpose of comparing relative rates of regeneration
of the different parts, especially the rate of regeneration of the
appendages as compared with that of the eyes.

The experiments upon the eyes consisted in either the removal
of a part of the eye or of the whole eye. ‘The part removed varied
greatly in the different series of experiments and more or less in
individuals of the same series. A limited number of experiments
upon Palzemonetes included the removal of both eyes or the
removal of one eye with a part of the brain; these operations in
most cases resulted fatally. The effect of splitting the eye was

166 Mary Isabelle Steele

also tried upon two series of Palamonetes, each series was com-
posed of a considerable number of individuals. Results, however,
were not particularly different from those obtained after removing
a part of the eye.

A total of 600 Palamonetes and hermit crabs had either one or
both eyes operated upon. A much smaller number of Crangon
and crayfish were used. No accurate account of the Ascellus,
Oniscus and Gammarus was kept. More than 50 per cent of the
Palamonetes and hermit crabs died immediately, or within a
short time, after the operation, many of them dying within a few
minutes after the eye was injured. Of the survivors about 58 per
cent lived through one or more moults.

Forty-two Crangon had the eye operated upon and of these one
died of the operation. The crayfish used were for the most part
C. gracilis, measuring from 12 to 15 mm. in length, that had
moulted but once after hatching.

Palamonetes and Crangon moult once about every ten days to
three weeks. The hermit crabs moult much less frequently, often
but once in two or three months. The hermit crabs regenerate,
however, as rapidly as Palemonetes or Crangon.

Considerable difficulty was experienced in keeping the animals
alive and in keeping individual records. Finally the plan of
keeping each animal in a separate finger bowl was adopted. This
method was fairly satisfactory except in very warm weather.
Then the water became warm and unless it was changed often the
animals soon died. Chopped-up bits of clam were fed to them
two or three times a week. Great care had to be used in warm
weather for the water became foul, if the food was left in more
than three or four hours, and caused the death of the animals.

In spite of all precautions various accidents occurred which
resulted in the death of promising material. Twice attempts
were made to transfer the experiments from Philadelphia to
Woods Hole or vice versa with disastrous results in each case.
The failure was due in part no doubt to the extreme warm weather.
For although every known precaution was taken most of the
animals died within twenty-four hours.

A number of the ordinary fixing fluids were used to preserve

Regeneration in Compound Eyes of Crustacea 167

material. Among those most frequently used were Fleming’s
osmic fixative, Perenyi’s fluid, Kleinenberg’s picro-sulphuric,
picro-aceto-sulphuric, Petrunkewitsch’s fluid and alcohol acetic.
Other fluids also were used and boiling water was tried. ‘The
best results were obtained from picro-aceto-sulphuric and Perenyi’s
fluid. In any case it is difficult to obtain a fixative that does not
shrink the inner tissues from the chitin, the regenerated tissues
being much more easily affected in this respect than normal
tissues.

The embedding was done altogether in hard parafiine 54° to
58° C. melting point. It was necessary to embed the material for
a long time in order to cut it without tearing the chitin from the
softer tissues. [he most satisfactory infiltration was obtained by
placing the objects first in equal parts of oil and paraffine, leaving
them on top of the water bath over night and then replacing the
oil and paraffine with pure parafiine, leaving them on top of the
water bath from eight to ten hours longer. Finally they were put
in the parafiine bath from one to two hours. Even after the most
thorough infiltration it was well nigh impossible to obtain complete
series of good sections, because of the difficulty of cutting through
the different textures of the material. In dehydrating preparatory
to embedding, cedar or bergamot oil was usedin preference to xylol
as these oils made the tissues less brittle.

The chief stains employed were Fleming’s triple stain and
Heidenhain’s iron hematoxylin. Various counter stains were
used with the iron hematoxylin but the most generally satisfactory
were acid fuchsin and orange G.

In most instances no attempt was made either to soften the
chitin or to remove the pigment before the eyes were sectioned.
As a rule, however, the material was fixed shortly after a moult so
that the chitin was as soft as could be obtained. Any sort of a
reagent used to soften the chitin seemed to be more or less inju-
rious to the softer parts, particularly the regenerating tissue.
When the chitin was removed, however, 2 per cent nitric acid in
70 per cent alcohol was found to be most satisfactory. It was
generally disadvantageous to remove the pigment because this
destroyed many of the landmarks both in respect to the regener-

168 Mary Isabelle Steele

ating tissue and the condition of the remaining old parts. Sections
were sometimes depigmented on the slide. For this Mayer’s
chlorine method was used.

Table I gives in brief a record of the individuals operated upon,
the character of the operation and the end results.

III Tur Normat Aputt EYE

Before taking up the discussion of the regeneration of the eye
it will be perhaps well to give a brief description of the structure of

Text Fig. 1 The lines, ab, a-c, a-d, a-e, a-f, a-~g and a-h represent approximately the different
levels at which a part or whole of the eye has been removed. When the cut was at level as low as a-e
a part of the optic ganglion was usually involved. In Palemonetes the eye never regenerated when the
cut came as low as the level a-e. Hermit crabs may regenerate an eye from the level a-f and a hetero-
morphic appendage from the level a-h.

the normal eye. The nature and extent of the operations and the
subsequent changes in the eyes will then be more easily under-
stood. The eye of Palamonetes has been concisely described by
Parker (’97) and the following description is adapted from his.
It is to be noted that the eye structure of the three forms experi-
mented upon is practically the same.

Regeneration in Compound Eyes of Crustacea

169

TABLE I.
PALAEMONETES
| No. of | Died Lived | Showed
Series | individ- Operation | Date from and _regenera-
| uals | operation| moulted | tion
I 16 part of cornea removed May 10 12 4 °
TS || £2. part of cornea removed july II 8 bs I
Ti 38 part of cornea removed July 20 29 3 | 8
IV 40 part of cornea removed July 28 28 II 8
Vv 15 part of cornea removed July 30 3 12 °
VI 16 part of cornea removed August I 12 z: I
VII 15__| partof cornea removed August a 10 2 3
Vill 12 part of cornea removed November 5 5 5 °
Ix ez 7am part of cornea removed | November 9 8 9 °
ibe ||) 4G entire cornea removed | March 10 4 7 °
|) 38 entire cornea removed July 20 8 9 I
Ty, || 30 entire cornea removed July 29 28 2) °
v | 30 entire cornea removed July 30 20 9 °
VI 13 entire cornea removed August 4 8 5 °
VII 12 entire cornea removed December 30 II I °
entire eye removed | |

I 12 both eyes | January I 4 6 °
II 12 entire eye | March 5 9 Hie | °
100% 1) ae both eyes March 5 12 On eo
DV; 18 both eyes March 10 10 5 °
Vv 45 entire eye April 19 12 20 °

I 20 eye split March 5 5 12 3

I 28 eye split May 24 nO) Ee | °

|
HERMIT CRABS

ee | ara, | cornea removed March 25 2 8 | 3

I 12 | cornearemoved May 26 2 7 | 2
TIL 25 | cornearemoved July 9 2) eer O nel em nis
I 15 entire eye removed | May 26 I | 72.) | I

I 25 entire eye removed July 9 | Aan era 9
Ut 8 entire eye removed October 16 oe || fo)
IV 12 entire eye removed November 27 | 2 6 | )

CRANGON
Ty) 20 part of cornea removed August 4 | ° 19 10
rE 22 eye removed August 4 | I 19 I

Note.—Removing the entire corneal portion of any of the forms was always accompanied by the
removal of part of the optic ganglion, In many cases as much as half of it. It frequently resulted
that all of the corneal portion subsequently degenerated after a part of it had been removed, and it
also frequently happened that the removal of the entire corneal portion resulted in the loss of the
whole eye stalk, consequently the above table can be used only as a very general indication of the
character of the operation.

170 Mary Isabelle Steele

The compound eye may be regarded as that part of the optic
apparatus contained in the eye stalk. It consists of a large num-
ber of ommatidia occupying the distal end of the stalk and a
series of four ganglia which extend through the axial portion of the
stalk. This series of ganglia for present purposes may be regarded
as a compound ganglion composed of four rather distinct sections
or ganglionic masses united to each other by nerve fibers. The
ommatidia are connected with the distal section of the optic
ganglion by the retinular nerve fibers. ‘The optic nerve passes
inward from the proximal section of the ganglion to unite the eye
with the brain. “The basement membrane forms a sort of partition
between the ommatidia and the optic ganglion. ‘The transparent
chitinous covering over the ommatidial region is known as the
cornea.

Each ommatidium is composed of the following cells: two cor-
neal hypodermal cells, four cone cells, two distal retinular cells,
eight proximal retinular cells one of which is rudimentary, and a
variable but small number of accessory pigment cells. Black
pigment granules are contained in both proximal and distal
retinule and are found only in these cells. “Uhe yellowish pigment
is confined exclusively to the accessory pigment cells. “The differ-
ent cells enumerated above give rise to the structures that consti-
tute a complete ommatidium. ‘The two corneal hypodermal cells
secrete the square corneal facet which covers the outer surface of
the ommatidium. Immediately beneath the corneal hypodermal
cells is the crystalline cone formed by the four cone cells. The
nuclei of these cells are located in their distal ends. ‘The main
body of the cone appears as a dense hyaline secretion. Proximally
the cone is less dense in structure and tapers to a slender stalk
lying between the coneandrhabdom. ‘The rhabdom, according to
my observations, is a swollen spindle-shaped structure proximal to
the inner ends of the cone cells. ‘The distal retinular cells lie near
the inner end of the cone; the proximal retinule surround the
distal end of the rhabdom. ‘The proximal processes of the retin-
ulz extend over the rhabdom and pass through the basement
membrane as the retinular nerve fibers to enter the optic ganglion
below. The accessory pigment cells lie both above and below the
basement membrane.

Regeneration in Compound Eyes of Crustacea 7

Before closing the description of the normal eye mention should
be made of another point. In young individuals in each of the
species examined in this series of experiments there is present a
growth zone in the ommatidial region. From this zone the num-
ber of ommatidia is increased as the animal grows older and
increases in size. In longitudinal sections cut in a horizontal
plane this zone is apparent on the inner edge of the eye as a narrow
band of elongated cells situated above the basement membrane
and between it and the completely developed ommatidia. In
some instances partially differentiated ommatidia can be recog-
nized in this growth zone. This zone has been mentioned by
Parker and others. It is briefly described in this connection now
because it is probable that in some instances ommatidia that have
apparently regenerated have in reality developed from the growth
zone.

The terminology used in the above description and in the dis-
cussion of the regenerating eye is that used by Parker. ‘The
series of optic ganglia described by him as occupying the eye stalk,
however, have been referred to in this paper as different divisions
of a single ganglion, it being thought that the matter could be
treated with less confusion in this way. Also, the distal portion
of the eye stalk, called the retina by Parker, is referred to here as
the ommatidial portion. It is composed of a large number of
individual ommatidia and the more general use of the term,
retina, does not imply all the structures composing the ommatidia.
The structure of the rhabdom as described in the present paper
is not in full accord with Parker’s description.

While the above description applies especially to Palazmonetes
it is sufficiently accurate for the other forms described in this work
to serve all purposes. The most marked differences in the struc-
tures of the eye of Palamonetes and of the other forms used are as
follows: In Crangon and in crayfish the eye stalk is much shorter
and proportionately greater in diameter than in Palamonetes.
Also, in Crangon some small glands are found located just below
the basement membrane. Hermit crabs have long slender eye
stalks similar to those of Palamonetes except that at the base and
occupying the dorsal inner edge there is a small pointed squame

172 Mary Tsabelle Steele

bearing a number of sensory hairs. (Fig. 37, 0-5q.) And finally,
there are no accessory pigment cells in the hermit crab’s eye.
These cells are very conspicuous in the eyes of Palamonetes.

IV THe PRELIMINARY REGENERATIVE PROCESS

The preparatory stages leading to the regeneration of an eye
stump consist chiefly of the following processes; the healing of the
wound, the removal of the injured tissues and the active prolifera-
tion of new cells of a comparatively undifferentiated character.
In removing any part of the eye the injury to the remaining soft,
inner tissues is considerable. Especially is this true when the cut
passes through the ommatidial region. Much of the tissue sur-
rounding the wound is crushed and torn out of place. On this
account the process of healing over the cut surface is much more
difficult to follow than the healing of the wound after an antenna or
leg has been removed.

Before taking up the description of the preliminary regenerative
process it will be perhaps of interest to give in brief the immediate
effects of the operation. The death of the animal which so fre-
quently follows close upon the operation seems often to be due
chiefly to nervous shock. It cannot be caused by loss of blood
alone for usually there is no profuse bleeding. When the eye of
Palzmonetes is operated upon the animal often turns over and
over ten to forty times as soon as it is released and returned to the
water. Many of the animals die before they succeed in nghting
themselves. Others lie upon their sides several hours after they
have ceased revolving and die without showing any normal activi-
ties or regaining their equilibrium. It very seldom happens that
animals which whirl over and over many times after the operation
ever recover from its immediate effects. “These apparently help-
less motions indicate that the operation has caused the loss of
equilibrium.

Crayfish sometimes exhibit these uncontrolled whirling move-
ments. Similar movements are also noted in fresh-water Gam-
marus and in Ascellus. In Gammarus and Ascellus these move-
ments are executed after the removal of the antennz or some of

Regeneration in Compound Eyes of Crustacea 173

the mouth parts. This shows that the effects are not specific-
ally connected with operations upon the eye. Whatever its ini-
tial cause the effect is transmitted to the whole nervous system.

In many cases the operation seemed to affect the animal more
seriously when only the upper part of the eye was removed than
when the entire eye was cut off. Frequently the relative number
of survivors was greater in the latter than in the former case. In
other instances the animal did not seem greatly affected by the
operation regardless of whether the whole or a part of the eye was
removed. (See Table I.)

The immediate visible effects upon the eye may be briefly
described as follows. As soon as any part of the corneal covering
is removed or even as soon as a rent is made in it a considerable
amount of the soft, viscous, inner tissue flows out through the
opening. It is perhaps carried out by the escaping blood. Much
of the pigmented, retinular tissue seems to escape, perhaps because
it is softer and more viscous than the other tissues. After an hour
or so the remaining, inner tissues are seen bulging out and above
the general level of the surface. This is probably on account of
the destruction of the normal tension of the tissues due to the
changed pressure conditions at the wounded surface. A similar
appearance is obtained when the surface injury consists of a rent
torn in the cornea with a needle.

A HEALING OF THE WOUND AND FORMATION OF NEW CUTICLE

In a few hours after the operation most of the pigmented tissues
have disappeared from the surface of the wound and the swollen
surface takes on a whitish appearance. ‘This white swollen surface
is apparent for several days. Not until the fourth or fifth day 1s
there any sign of the characteristic red-brown crust which generally
forms over wounds in Crustacea.

Sections of an eye fixed six and a half hours after the operation
show no definite indications of the healing of the wound. A
great deal of the broken and mangled tissue lies outside the wound
and hanging to the cuticle about its edges. Inside, the tissues are
twisted and misshapen. At the edges of the wound there are

174 Mary Isabelle Steele

slight indications that it is preparing to close over. But the
quantity of material lying inside and out makes it impossible to
determine what tissues are taking part in closing the wound.

During the next twelve or fifteen hours the changes are still not
clearly defined. ‘The interior still presents a rather badly confused
mass of injured tissue. Near the edges of the wound, however,
there are evidences that the hypodermis has begun to push outward
to cover the cut surface. For the most part ‘the wound shows a
smooth even surface which indicates that the passage outward of
the injured tissues has ceased and that a sort of equilibrium had
been established. The mass of tissue closing the cut seems to be
made up of a few hypodermal cells and cytoplasmic strands, a
considerable accumulation of blood cells and the nuclei of the
breaking down tissues of the eye. Around the edges of the cut
occasional strands of hypodermis with a very few nuclei can be
distinguished.

Sections of an eye fixed about forty hours after the operation
show the beginning of crust formation. Almost the whole surface
has been covered. Judging by the reaction to stains, the part
which may be considered the matrix of the crust is formed by an
attenuated, chitinous secretion of the hypodermis. In this matrix
are embedded numerous nuclei of the injured tissues together with
a great many blood cells and a few hypodermal cells. In some
parts the crust is sharply marked off from the underlying tissues
by a space filled with coagulated plasma. Over one part of the
wound the crust is not yet fully formed. At this point hypodermal
strands containing elongated flattened nuclei are seen stretched
across the space still uncovered. The strands appear in two or
three layers with very few nuclei in each layer. No definite
centers of cell proliferation can be recognized at this time.

After the crust has covered the cut surface it continues to increase
in thickness for two or three days, then hardens, turns a bright
reddish brown color and remains over the stump until a moult has
occurred. ‘The crust takes no further active part in the healing
and regenerative processes. Fig. 46 represents in a semidia-
grammatic manner the crust formed over the wound in a crayfish
eye about sixty hours after the operation. ‘The crust is continu-

Regeneration in Compound Eyes of Crustacea 175

ous with the inner surface of the cuticle covering the rest of the
eye. There are still masses of the injured inner tissues that have
been excluded by the formation of the crust clinging to its outer
surface. ‘The old tissues in the interior of the eye stump have
shrunk back from the crust leaving a considerable space occupied
by coagulated plasma. ‘The old cuticle and the matrix of the
crust both stain deeply either with orange G or acid fuchsin.

No distinct cuticle can be recognized for several days, from six
to eight, after the operation. In the eyes of Palamonetes that
have moulted seven or eight days after the infliction of the injury
a cuticle which corresponds approximately in thickness with the
cuticle covering the remainder of the eye has formed over the
wound. ‘This new.cuticle is much looser in texture than the old
cuticle. Regeneration does not take place so rapidly in crayfish as
in the marine forms examined so that a new cuticle is somewhat
longer in forming. Frequently a considerable space intervenes
between the overlying crust and the cuticle which has formed
beneath it. This is probably due to the recovery of the tissues of
the stump from their early swollen condition during which time
they were gorged with blood and occupied more than their normal
amount of space. It is not unusual to find considerable spaces
between different layers of the new cuticle as if a shrinking of the
tissues had taken place during the process of forming the new
layers of cuticle. The shrinking of the interior tissues without
doubt also accounts in part for the folds and wrinkles which
often appear in the cuticle over the wound.

The secretion of the new cuticle which grows over the wounded
surface begins some little distance back from the cut edges of the
old cuticle and is continuous with its inner layers. Fig. 47 1s a
semidiagrammatic representation of the relation of the old and the
new cuticle and the exclusion of the broken down tissue by the
development of the new cuticle beneath it. Only eight nuclei
appeared beneath the new cuticle in the section as shown in Fig.
47. This figure is taken from an eye that had been injured by
tearing through the cuticle with a needle. Reference to the figure
shows that very little of the old cuticle had been removed. Fig. 50
shows part of a section near the edge of the wound, Fig. 51 part of a

176 Mary Isabelle Steele

section near the center of the wound. Both figures show the
distribution of the hypodermal nuclei beneath the new cuticle that
had formed over the cut surface of the eye of a Palamonetes ten
days after the operation. Examination of Figs. 47, 51 will show
that a new cuticle may be secreted before a complete hypodermis
can be recognized.

Recently hatched Cambarus gracilis, 12 to 15 mm. in length
had eyes injured by tearing through the cornea with a sterilized
needle. The eyes operated upon in this manner were fixed at
different times, varying from eleven to thirty-five days. All of
the eyes, however, were fixed before a moult had taken place. In
this way it was possible to determine the precise position of the
original injury. Sections of such eyes show one point conclusively,
at least for Cambarus gracilis. ‘That is that the proliferation of
new cells begins from the hypodermis immediately surrounding
the rent. From the edges of this proliferating center new cells
push out to replace the cells that were removed or that have broken
down. Previous observations made upon the regeneration of the
eye in crayfish (Steele ’04) indicate that crayfish probably do not
regenerate a functional eye. It appears, however, that the prelim-
inary regenerative processes are essentially the same in crayfish as
in the other forms examined.

It is frequently the case that a much greater proportion of the
soft inner tissues are destroyed than of the outer cornea or the
hypodermal layer beneath it. When the cut is made the retinule
and the lower ends of the cone cells press out of the wound and
leave the outer ends of the cones and the hypodermis practically
undisturbed. Such a condition is particularly noticeable in eyes
that were operated upon by tearing the cornea with a needle. In
such cases the hypodermal cells 1m situ secrete the new cuticle.
This cuticle is, however, without corneal facets, a fact which
shows that while the operation neither removed nor caused the
disintegration of the hypodermal cells it still affected their activity
to such an extent that they no longer function in their usual
specialized manner. ‘They now function as the ordinary hypo-
dermal cells over the general surface of the body.

Sections frequently show a morphological transformation of the

Regeneration in Compound Eyes of Crustacea 177
Ps TEV

corneal hypodermal cells in situ that are engaged in the secretion
of the new cuticle. In the normal adult condition the pair of
corneal hypodermal cells that belong to each ommatidium appears
as much flattened cells crowded between the distal ends of the
cones and the corneal facets. Their nuclei stain faintly and
appear to be slender oval bodies lying flat against the cuticle. As
the distal ends of the cones in an injured eye break down the
nuclei of the corneal hypodermal cells enlarge, become rounded,
stain deeply and in every way show signs of increased activity.
Fig. 52 includes a series of figures showing the transformation of
the corneal hypodermal nuclei into the larger, more deeply staining
type seen in the regenerating eye. a and / inthis figure represent
the corneal hypodermal cells as they appear in the normal omma-
tidia. The other figures of this series, c,c’,d and e, show corneal
hypodermal cells, belonging to ommatidia that have degenerated
either wholly or paral oie c, c’, e the distal ends of “he cones
still remain almost intact and in c the nuclei of the corneal hypo-
dermal cells appear but little larger than those associated with nor-
mal ommatidia. In c’ and e, however, the nuclei of the corneal
hypodermal cells are much enlarged, stain deeply and the cyto-
plasm surrounding them appears granular and loosely reticular.
One nucleus to the left of the figure in d appears to be preparing to
divide amitotically. That the nuclei shown in d are the trans-
formed nuclei of the original corneal hypodermal cells is deter-
mined by the fact that on either side of these nuclei are others still
associated with partially disintegrated cones. Their original
character is also suggested by the fact that they are grouped in
pairs. A regenerated, rather than a transformed, hypodermis
over the ommatidial region never shows the nuclei arranged in
pairs in the early regenerative stages. “That the new cuticle has
been secreted by ieee Paneionued hypodermal cells is shown by
the relations of the two structures. The cytoplasmic strands of
the hypodermis are continuous with the inner layers of the cuticle
(Fig. 52d).

Of course it is not absolutely proved that the transformed
hypodermal cells take part in the later regenerative processes.
This could not be done without examining a very great number of

178 Mary Isabelle Steele

stages. But even the examination of a series of sections from the
same eye will show that the indications are strongly in favor of the
view that these hypodermal cells remain active and constitute the
hypodermis during the subsequent regenerative processes. Fig.
53 1s taken from the same eye as d in the series shown in Fig. 52.
In the later figure it is evident that regeneration is in progress.
The hypodermal cells, however, show a tendency toward a paired
arrangement indicating that they are the original hypodermal
cells.

After a part of the corneal covering has been removed it is
evident that an entirely new hypodermis must be regenerated over
the wounded surface. In crayfish it was seen that active cell
proliferation began near the edges of injured hypodermis and that
new cells pushed outward from these centers. ‘The early stages
have been examined in a number of eyes of Palemonetes. The
centers of cell proliferation in this form are not so apparent. The
nuclei which in the early stages appear beneath the cuticle that
covers the wound are very few and lie far apart. “Their number
increases not chiefly by the repeated multiplication of nuclei at the
edges of the wound but by the repeated division of the nuclei that
are pushed out onto the wounded surface. During these early
stages while the nuclei are actively dividing the cytoplasm is very
loose and reticular and the cell boundaries are indistinguishable.

In many cases the new cuticle which becomes apparent after the
first moult is somewhat definitely separated into a dense outer
portion and a semifibrous inner division. The inner division
often appears as an interlacing network of fine fibers, many of
which can be traced into the hypodermis beneath (Fig. 53). The
loose incompact character of the hypodermis over the wounded
surface probably indicates a high degree of activity of the hypo-
dermis in this region.

It is apparent from the foregoing description of the formation
of the hypodermis covering the wound that the regenerated hypo-
dermal cells may arise in two ways. They may arise by a trans-
formation of the old corneal hypodermal cells im situ in which
they assume a less specialized réle or they may arise by the migra-
tion of a limited number of hypodermal cells from the edges of the

Regeneration in Compound E yes of Crustacea 179

cut, which later multiply until a complete hypodermis is formed.
In either case the first new nuclei must be contributed by the
remaining hypodermal cells. Whenever the cut has not removed
the entire ommatidial portion the remaining corneal hypodermal
cells must assume a somewhat less specialized role in order to
form the first new nuclei of the regenerated hypodermis.

B REMOVAL OF THE INJURED TISSUE

The fact that the inner tissues of the eye are so much softer than
the chitinous outer covering renders it impossible to operate upon
the eye in any way or to remove any part of it without serious
injury to the remaining softer tissues. It is evident that, before
any considerable regeneration can take place, the injured tissues
must be either repaired or removed. All the observations made
upon regenerating eyes tends to show that none of the injured
tissues except the hypodermis ever repair themselves.

Sections of an eye fixed six and one-half hours after the operation
show that considerable changes have already taken place in the
injured tissues. The effect is particularly noticeable in the
retinula. Many of the retinular nuclei have become separated
from the pigmented retinular processes and appear as rounded
bodies surrounded by a dense mass of cytoplasm. ‘These are
irregularly scattered among the other tissues. Some parts of the
interior have become fairly clear of the broken down structures
and are occupied chiefly by coagulated plasma. In other parts
of the eye the injured tissues lie in confused masses.

The changes in the next twelve or fifteen hours do not show
much advance over the earlier stages. ‘The interior still presents
a badly confused mass of broken down tissue. In some parts,
however, the cone nuclei appear larger, the bodies of the cones
have begun to dissolve and the number of rounded retinular cells
appear somewhat more numerous than in the earlier stages, their
nuclei showing irregularities in shape (Fig. 75a and b).

During the earliest stages, six to sixty hours after the operation,
the ommatidia that are still intact always appear bent and twisted
out of their normal positions. Later stages show that these

180 Mary Isabelle Steele :

uninjured ommatidia have regained their original shape and
position. ‘This temporary contortion of the ommatidia seems to
be due to the immediate effects of the operation, reduction of
pressure, destruction of normal tension relations, etc. That in
the later stages they appear normal again indicates that they have
adjusted themselves to the new conditions imposed upon them by
the operation.

Besides those ommatidia that are actually injured by the
operation a large portion of those remaining frequently degenerate.
The destruction of the tissues of the eye is, consequently, much
more extensive than the original injury. ‘This fact is strikingly
illustrated by eyes in which the original injury consisted in thrust-
ing a needle into the ommatidial portion. In several such in-
stances the entire, or almost the entire, eye has degenerated.
Instances of this kind have been observed in the eyes of sev-
eral Palamonetes and also in the eyes of young Cambarus gracilis.
Similar phenomena have been observed in eyes of fresh water
Gammarus. To be sure the eyes of Gammarus are quite small
which may account in a measure for the fact that in six or eight
eyes examined in serial sections only one showed any of the old
ommatidia intact. In Cambarus gracilis there were two instances
out of six in which none of the ommatidial portion remained. In
one of the cases the entire eye had degenerated; not even the vestige
of the stalk remained. All of this degeneration took place in
about thirty days and without a moult. In the other case all of
the ommatidial portion and more than half of the optic ganglion
had degenerated during the same period. In other eyes of the
same series very little degeneration followed the operation. In
these extreme cases it seems probable that some infection played
a part.

In the degeneration and removal of the injured tissues the
retinula) degenerate most rapidly. In many cases they break
down within the first few hours after the operation. ‘Their long
pigmented processes become separated from the cell body and
collapse into shapeless masses of pigment, which become scattered
through the other tissues. The greater part of this pigment
finally gathers in clumps near the level of the basement mem-

Regeneration in Compound Eyes of Crustacea 181

brane. Although the retinulze are the first to collapse their
remains are the last to be gotten rid of. Evidently the pigment is
absorbed and removed only with difficulty. Often in regenerating
eyes that have completely regenerated new ommatidia much of the
old pigment remains.

The cell bodies of the retinulz after losing their pigmented
processes appear as large nuclei surrounded by a narrow zone of
condensed cytoplasm. Within a short time these cells become
scattered widely through the eye. After a few days their nuclei
appear irregularly shaped and soon afterward become conspicu-
ously polymorphic (Fig. 75a and b). In the usual course of
events these retinular cells disintegrate and disappear. But under
some conditions they apparently remain and later multiply and
give rise to an abnormal development of tissue that secretes pig-
ment.

The rhabdoms and the inner ends of the cones also degenerate
within a short time after the retinula. The cones continue to dis-
solve from the proximal ends distally. The last parts to disappear
are the outer ends in which are embedded the cone nuclei. Before
the dissolution of the cones is complete the cone nuclei appear
greatly enlarged and stain deeply. Their enlarged appearance is
probably largely due to the disintegration of the cone substance
from about them.

As the disintegration of the tissues proceeds the interior of the
eye becomes filled with a granular mass containing scattered
nuclei and masses of pigment. This granular mass which is
usually very much vacuolated is made up of the remains of the old
tissues together with more or less of coagulated plasma and blood
cells. Sometimes the remains of the old cones appear as long
tapering bands of granular material extending from the periphery
inward.

Fig. 51 represents a small area of the disintegrated ommatidial
structures as it appeared in the eye of Palamonetes ten days after
the operation. Only two or three nuclei lie close beneath the
cuticle and a few others lie scattered deeper in. Most if not all of
these more deeply located nuclei are the remains of the old omma-
tidial structures. In the lower part of the figure are some old

182 Mary Isabelle Steele

pigment remains. ‘The granular mass occupying the greater
part of the figure is made up chiefly of partially dissolved cones.
The slender strands which can be traced through the granular
mass are in part at least made up of new cytoplasm. At certain
points the cytoplasmic strands are seen to be continuous with the
inner layers of the cuticle.

Fig. 48 is taken from a section of an eye of Palamonetes seven
days after the removal of part of the ommatidia. A surface view
from which this figure is taken is shown in Fig. 1. Practically all
the material represented in Fig. 48 except the cuticle is made up of
disintegrated ommatidia. The long band extending inward
shows approximately the position of a former cone. ‘The large
vacuolate spaces in the upper part of the figure are old nuclear
remains. In the process of disintegration the nuclei at first
enlarge and stain deeply. Later the nuclear contents disappear
although the nuclear membrane persists for some time longer,
often becoming shrunken and folded into a variety of shapes.

It has been seen that the disintegration of the injured tissues
begins immediately after the operation and that the greater part is
accomplished in from one to three days. ‘The distal ends of the
cones alone remain intact for a much longer time, in some cases
from two to three weeks and occasionally even longer. ‘The
removal of these disintegrated tissues is much slower than their
dissolution.

Regeneration proper may and usually does begin within a few
days after the old structures have broken down and progresses
simultaneously with their removal (Figs. 49,50). One part of the
eye may show ommatidia differentiating while another region is
still occupied by disintegrated old structures. The individual
differences in the rate of regeneration of such frequent occurrence
is probably largely dependent upon the variations in the length of
time required for the removal of the old structures. This prob-
ably also accounts for the fact that regeneration does not take place
uniformly throughout the same eye. ‘The part of the eye that gets
rid of the injured tissue soonest regenerates first.

The above observations apply in general to all the forms used.
Crangon, however, offers a significant exception in that the injured

Regeneration in Compound Eyes of Crustacea 183

tissues disintegrate much more slowly than in Palzmonetes, her-
mit crabs or crayfish.

C METHOD OF CELL DIVISION

In the earlier preliminary stages of the regenerative processes
it is impossible to distinguish cell outlines. We should there-
fore speak of nuclear divisions, perhaps, instead of cell divisions.
In later stages the cytoplasm becomes differentiated about the
individual nuclei. In all cases of the regeneration of the eye the
nuclei are increased by amitotic division. Before a definite
hypodermis is established the nuclei can be seen in various stages
of constriction, separating off new nuclei for the development of
the future underlying structures. ‘There seems to be no perfectly
definite manner in which the constriction and separation of a
nucleus into two parts takes place. One or two characteristic
forms, however, appear so frequently as to be readily distinguished.
The two most usual types are seen in the three nuclei occupying
the extreme right of Fig. 53. When a nucleus divides in a plane
parallel to its long axis it usually assumes the form of the outer one
of the three referred to. The formation of the notch on one side
gives the nucleus a peculiar heart-shaped appearance which seems
characteristic and is easily recognized. Figs. 49 and 53 show
nuclei of this same type further advanced in division. ‘The other
type referred to is represented by the other two nuclei of the three
at the extreme right of Fig. 53. Apparently these two nuclei were
recently formed by the longitudinal division of one of the heart-
shaped nuclei, like the one lying beside them. Each of these two
is now dividing unequally by a transverse constriction.

A dividing nucleolus can sometimes be seen but more frequently
a definite nucleolus cannot be distinguished. When nucleoli are
seen in dividing nuclei they usually appear with a darkly staining
strand of material connecting them. In sections of young Cam-
barus gracilis eye, nine and sixteen days after the operation, two
nucleoli can be recognized in many of the nuclei. In those nuclei
that are dividing one nucleolus lies in each part. In all except in
the later stages of regeneration of the eye nuclei dividing amitot-

184 Mary Isabelle Steele

ically can be found in great abundance. But at no time are
mitotic divisions seen. In a careful examination of a large num-
ber of sections only one cell has been found that suggested the
possibility of its being in mitotic division. ‘The appearance of
this one suggests a late anaphase or an early telephase. Conse-
quently it is not certain that this is mitotic division. “This nucleus
appears near the left edge of Fig. 53 at K. In any case it must be
admitted that amitosis is the regular method of cell division in
regenerating eyes of the forms studied, since in every specimen
examined during the stages of cell divisions amitosis has been
observed and mitosis has not been seen.

That such is the case is somewhat unexpected since Miss Reed
(o4) found mitotic division abundant in the regenerating leg of a
crayfish. Miss Reed, however, observed that there were no
mitotic figures during the early stages of regeneration, although
new cells were being eapily formed. Perhaps we may infer from
this that amitosis took place in the regenerating leg of the crayfish
during the preparatory stages at ee But one would hardly
expect such differences in cell division in forms so closely related
for example as crayfish and Palemonetes. No observations have
been made upon histogenesis in the regenerating appendages of
hermit crabs, Palemonetes or Crangon. Hence it cannot be
said whether or not the eye furnishes an unique exception to the
regeneration of other parts in these forms.

Recently, however, many have come to regard amitosis as a
phenomenon of more frequent occurrence than has been generally
supposed. Meves (’g1 and ’94), McGregor (’99), Child (’04 and
’07), all describe amitosis as a normal phenomenon and con-
sequently no longer accept Vom Rath’s view that a cell is nearing
its final dissolution when it begins to divide amitotically.

On the other hand many of the more conservative investigators
are unwilling to admit that amitosis occurs as a normal phenom-
enon and believe that the apparent cases of amitosis can be
explained on some other grounds. But in all the instances hitherto
described amitosis has been found occurring along with mitosis.
In the present case, however, all of the cell divisions are amitotic
and they all take place in cells derived from the hypodermis; in

Regeneration in Compound Eyes of Crustacea 185

these respects the regenerating eyes investigated in this series of
experiments offer a unique instance in their method of cell division.

V REGENERATION OcCURRING AFTER DESTRUCTION OF
DistaL Part oF EYE

Under this heading will be discussed the results obtained from
eyes injured in varying degrees but not exceeding the destruction
of more than the distal two sections of the optic ganglion. ‘The
injury originally inflicted varied from tearing the surface of the
cornea with a needle to cutting off the whole top of the eye so that
at least the first part of the second section of the optic ganglion had
been removed. Frequently, however, as has been explained in
Section IV the part of the eye ultimately lost was much greater than
that originally removed. For in cutting the eye with scissors or
tearing it with a needle much of the tissue surrounding and under-
lying the wound was so injured that it afterward degenerated.

In the series of experiments to be described under this division
nearly two hundred Palamonetes, twenty Crangon and fifty
hermit crabs were used. Fifty per cent of the Palamonetes, 5 per
cent of the Crangon and Io per cent of the hermit crabs died of the
operation. Of those that survived the loss of blood and the
nervous shock of the operation a considerable number died from
other causes without having undergone a moult. Forty-five
Palamonetes, seventeen Crangon and twenty-nine hermit crabs,
however, lived through at least one moult. Among these a num-
ber showed no regeneration either from surface examinations or
from sections. The greater number of those that gave surface
indications of regeneration and for comparison a number that
showed no regeneration have been sectioned and examined.

A REGENERATION OF THE FUNCTIONAL EYE

Experience has shown that the number of days an experiment
covered serves to indicate only in the most general way the stage
of regeneration. While the time element naturally constitutes a
most important factor the rate of regeneration is also dependent
upon the season of the year, the age and the physiological activity

186 Mary Isabelle Steele

of the individual and perhaps other factors not so apparent.
Besides individual differences displayed between members of the
same species there also appeared to be differences in the ability to
regenerate and the rate of regeneration. among the three species
chiefly used, these differences being much more marked in the
regeneration of an eye than in the regeneration of an appendage.
The hermit crabs seem to completely regenerate an eye in shorter
time than either Palamonetes or Crangon. But the final results
were similar in all three forms. The few significant differences
will be pointed out and discussed later.

rt Entire Preparations of Regenerating Eyes

A careful examination of the regenerating eye frequently reveals
a number of important features and is absolutely essential to the
later interpretation of the sections. Therefore outline surface
drawings have been made of all eyes later sectioned. In most
cases the normal eye was drawn in connection with the regener-
ating eye for comparison as to size, shape, etc. Frequently the
two eyes were sectioned together. These surface views which
had better be regarded as optical sections were drawn with a
camera after the eyes had been brought into oil preparatory to
embedding. Figs. 1 to 25 represent various stages of regenerating
eyes.

Fig. 1 shows dorsal and ventral views of a Palamonetes’ eye
seven days after an operation which removed the ventral corneal
surface and immediately after a moult. A comparison of the
dorsal and ventral views shows that the injury is confined chiefly
to the underside. In shape the eye is practically normal. It is
not even flattened on the underside as the thinning out of the
pigment near the center would seem to indicate. As is usually
the case the injured eye is much smaller than the uninjured one.
Not only the region operated upon but the whole eye decreases in
size after the operation. ‘This indicates that however localized
the operation may be the effect is much more extended. In this
particular case the part of the eye proximal to the injury measures
only three-fourths of the length of the same region in the unin-
jured eye.

Regeneration In Compound Eyes of Crustacea 187

Fig. 3 represents the ventral view of a Palamonetes eye ten days
after being injured. In this case also the injury extends across
the ventral side of the ommatidial portion. The pigment of the
broken down ommatidia can be seen scattered in flakes and
patches through the upper part of the eye. From the dorsal side
the eye appeared nearly normal but sections show that almost the
entire eye is in process of degeneration.

The specimen represented in Fig. 4 shows a regenerating eye
nineteen days after the removal of almost all of the ommatidial
portion. It is readily seen that very little remains except the eye
stalk. The pigment patches are remains of the original eye.
Across the end of the stump the cuticle is wrinkled and folded,
indicating that comparatively little new tissue has been formed and
that the cuticle follows more or less closely the rough uneven out-
lines of the wounded surface.

When the entire ommatidial portion has been removed or has
degenerated regeneration seems to be considerably slower than
when a large part of it remains uninjured. ‘The two specimens
shown in Figs. 9 and 1o afford a striking illustration of this fact.
Both of these eyes were operated upon at the same time. Each
animal moulted twice, the first time on the same day and the
second, a day apart. Both were fixed in picro-acetic at the same
time, thirty-two days after the operation. In Fig. g the injury
involved only the posterior ventral side, less than one-half of the
ommatidia. While in Fig. 10 the injury included all of the
structures lying distal to the basement membrane. Examination
of the sections shows new ommatidia completely differentiated in
Fig. 9 while in no part of Fig. 10 are they yet defined.

The next specimen, Fig. 20, presents a rather striking appearance
and suggests immediately that the regeneration taking place is not
altogether of the normal type. ‘This is a thirty-eight day specimen
and belongs to the same series of experiments as the preceding two
specimens. All of the part distal to the dark pigmented band is
regenerated tissue. [he pigment consists chiefly of the remains
of the old retinulz. It is evident even from a surface view that no
ommatidia have developed. Sections show, however, that on one
side the differentiation of cones is beginning (Fig. 69). Here it

188 Mary Isabelle Steele

may be well to mention a fact that has been observed a number of
times. The regeneration of the new ommatidia never presents a
uniform stage of differentiation in any case whether or not all of
the old ommatidia have disappeared. In fact it may be possible
to select several stages from the same eye and sometimes two or
three stages from the same section.

An eye thirty days after the injury, Fig. 6, shows some interesting
features in comparison with the one just described. From a
ventral view this eye shows no signs of remaining ommatidia and
from the dorsal side only a very few cones and facets are evident.
The upper part of the eye is transparent. Below this transparent
area are scattered patches of pigment representing the remains of
the old eye. Sections show that no new ommatidia have been
entirely differentiated, that the retinular cells have differentiated
and are establishing connections with the optic ganglion, that new
cone nuclei are being separated from the hypodermal nuclei, that
on one side a few of the old ommatidia remain and that in the
tissues lying nearest the old ommatidia new cones are being
developed. The most striking feature presented by this specimen
is the clearness with which the connections between the retinulze
and the ganglion cells can be made out. ‘These connections will be
discussed at length in the consideration of the detailed structures
of the regenerating eye.

Fig. 17 shows the ventral view of a regenerating Palamonetes
eye from a thirty-five day specimen. Examination of the dorsal
side shows that a large number of the ommatidia on that side
appear uninjured, the greater part of the injury being confined to
the ventral side as indicated by a surface examination. Sections
show, however, a gradation:of regeneration from a stage in which
there is no signs of cone differentiation to the complete formation
of a new ommatidium.

It was said above that hermit crabs regenerated an eye more
rapidly than either Crangon or Palamonetes even in instances
where a considerable portion of the optic ganglia had been
removed. Figs. 5, 12 to 15 show regenerated eyes of hermit crabs
after one or more sections of the optic ganglion have been de-
stroyed. Fig. 12 had at least the distal division of the ganglion

Regeneration in Compound Eyes of Crustacea 189

removed. ‘The regenerated eye shown in the figure was developed
within hive hrees days and after only one moult. This moult
occurred on the thirty-second day and the eye was fixed in Perenyi
the thirty-third day. Sections show that ommatidial structures
have been fully differentiated although incompletely developed.
One point is particularly noticeable in these sections; the omma-
tidia are very much shorter thaninthe normaleye. ‘This condition
was probably caused by the mechanical pressure of the covering
cuticle which forced the developing ommatidia into less space than
they would otherwise have occupied. Fig. 13 shows the eye of a
hermit crab regenerated from a stump in which not more than half
of the optic ganglion remains. ‘The regeneration in this eye took
place in thirty-eight days. One moult occurred twelve days after
the operation. Although extremely small the eye 1s practically
perfect except that the corneal facets have not yet developed.

Figs. 14 and 15 show two other regenerated eyes of hermit crabs
forty-one and sixty-seven days respectively after the operation.
Sections of the forty-one day eye do not show ommatidia as fully
developed as the thirty-eight day specimen previously described.
The sixty-seven day specimen shows the eye complete in all its
details even to the corneal facets. Whether a younger regenerated
eye might not show the corneal facets has not been determined
since no specimens were available between the forty-one day and
the sixty-seven day specimens.

A noticeable feature in all the regenerated eyes of the hermit
crabs is their small size in comparison with the normal eyes. It is
probable that the regenerated eyes would have increased in size
if the experiment had covered a longer period of time. Sections of
the eye shown in Fig. 12 indicate that the definitive size has not
been reached. For, lying outside the fully formed ommatidia are
others in the process of development. In the case of the eye
shown in Fig. 13 sections do not show any indications of partially
developed ommatidia and it may be that this eye would never have
reached the size of the normaleye. Perhaps this is what we should
expect since the amount of nervous tissue present is considerable
less than is normal.

It is more difficult to interpret the actual condition of a regener-

Igo Mary Tsabelle Steele

ating eye in Crangon than in either Palamonetes or hermit crabs.
The whole of the eye stalk in Crangon is thickly covered with
branching pigment cells. So that even after the eyes are brought
into oil very little can be seen in detail by examining them in toto.
Figs. 7 and 8 thirty-one and thirty-two days, respectively, are
fairly characteristic of the regenerating eye of Crangon. From
all that can be determined from the outside, regeneration seems
practically complete in each of these cases. Sections show,
however, that comparatively little regeneration has taken place.
A fuller discussion of the regenerating eye of Crangon will be taken
up elsewhere.

2 Details of Development of the Regenerating Ommatidia

The complete regeneration of the ommatidial portion of the eye
involves three stages. ‘These can be separated from each other
rather sharply although they overlap more or less. The first stage
consists in getting rid of the broken-down tissues and the healing
of the wound; second, the active proliferation of new cells; and
third, the differentiation of the new ommatidia. The first and
second stages have been discussed in Section IV.

a Regeneration of Retinulze

All the observations support the conclusion that the regenerated
ommatidia are derived entirely from the hypodermis. Before the
hypodermis covering the end of the stump has been clearly differen-
tiated, however, the proliferation of cells for the new structures
lying below has begun. So that at the same time hypodermal
cells are dividing in two planes, one at right angles to the periphery
to increase the number of hypodermal cells, and the other parallel
to the surface. The inner nuclei of the latter division migrate
inward and become the first retinular cells. As they migrate they
become elongated with their long axes radially arranged.

Fig. 54 shows the early stages of the separation of retinular
nuclei, some separating from the nuclei at the periphery and others
migrating in. At a comparatively early date these retinular
nuclei have migrated a considerable distance below the surface

Regeneration in Compound Eyes of Crustacea IgI

and may be seen in a relatively well defined row (Fig.68). Soon
after the retinular nuclei have been separated from the nuclei at
the surface they themselves begin to divide (Fig 68) in a plane at
right angles to their plane of original division. ‘These longitudinal
divisions may begin before the nuclei have reached their definitive
position. ‘This division continues until a band composed of many
nuclei has been formed. Figs. 60 and 62 show portions of such
bands.

Here and there single nuclei are found lying much nearer the
basement membrane than the retinular band (Figs. 60, 62).
These are occasionally seen constricted. It has been impossible
to determine with certainty the fate of these scattered nuclei but
there are evidences which suggest that they become the nuclei of
the accessory pigment cells.

The retinular nuclei even in the early stages of their migration
stain much more deeply than the nuclei at the periphery. But at
this stage no definite cytoplasmic outlines can be distinguished.
Very faintly staining delicate strands of cytoplasm, however, can
be found extending between the retinular nuclei and the periphery.
These strands form an intermingled network and with the nuclei lie
in a granular substance. Soon after the nuclei have reached their
definitive position the cell bodies of the retinule can be recog-
nized. Each nucleus appears surrounded by more or less definite
strands of cytoplasm which are radially arranged and extend
outward toward the periphery and inward to the basement mem-
brane (Figs. 60, 65). These can now be definitely recognized as
retinular cells. Usually it is easier to see the proximal than the
distal strands for at an early stage these lower processes begin to
secrete pigment and are consequently more conspicuous (Fig. 65).
At the stage represented by Fig. 62 there is only the merest begin-
ning of pigment deposition. ‘The fibers are but little differentiated
from their background which still seems to be composed largely of
a homogeneous granular material, probably to a great extent
coagulated plasma. :

At a stage such as 1s represented by Figs. 60, 62, in which there
is only the merest beginning of pigment secretion delicate cyto-
plasmic processes can be traced from the retinular nuclei inward

192 ; Mary Isabelle Steele

through the basement membrane beneath which they can be found
branching over the ganglion cells. Fig. 62 shows several isolated
retinular cells and their proximal processes which are seen extend-
ing through the basement membrane and reaching to the ganglion
cells beneath. There is no evidence that these retinular fibers are
directly connected with the ganglion cells; they seem merely to
twine around them.

When the fibers have reached the basement membrane they
may pass directly through it as the two shown in the left side of
Fig. 62, or they may extend along the upper face of the basement
membrane before entering the ganglionic mass below. ‘The
retinular processes frequently branch shortly before entering the
basement membrane or just as they emerge below it. No special
nerve methods were employed yet numbers of these fibers are seen
branching among the ganglion cells and many are readily traced
from the retinular nuclei to the basement membrane (Fig. 65).
It is only in very favorable specimens that the fibers can be traced,
however, through their whole length.

b Regeneration of the Crystalline Cones

Up to and including the stages shown in Fig. 65 and described
above there is no evidence of any differentiation of the crystalline
cones. A definite hypodermis, however, has been formed with an
increased number of nuclei, from which other nuclei are separating,
These are the cone nuclei (Fig. 63).

The formation of the cone nuclei does not appear to take place
in a uniform manner. The usual method, however, is by the
division of the hypodermal nuclei in a plane parallel to the periph-
ery, the inner nuclei thus formed being the cone nuclei. But
since in every ommatidium there are four cone nuclei and only two
hypodermal nuclei, it is evident that either the hypodermal cells
must divide twice or the first cone nuclei must themselves divide in
order to make the cone nuclei just twice the number of the hypo-
dermal nuclei. Observations indicate that in some cases the
second pair of cone nuclei arise by the division of the first pair.
In other cases, however, it is uncertain whether they arise in this
manner or whether they arise from the hypodermal nuclei. It is

Regeneration in Compound Eyes of Crustacea 193

probable, however, that they are formed by the division of the first
pair of cone nuclei as can be determined in some cases.

In the early stages of cone formation the inner surfaces of the
hypodermal cells lose their distinctive outlines. For at this time
there is no clear line of demarcation between the hypodermal and
cone cells. Fig. 63 represents one of the earliest recognizable
stages in crystalline cone formation. The hypodermal cells are
more or less definitely grouped into pairs and it is readily seen that
cone and hypodermal nuclei are not wholly separate. Extending
inward from the cone nuclei are very delicate strands of cyto-
plasm. ‘These strands seem to group the hypodermal nuclei into
pairs and by their branching and crossing form a much vacuolated
network.

Ata stage slightly more advanced (Fig. 64) the cytoplasm of the
cones has begun to assume a more definite cone shape. ‘There is
still, however, no distinct line of separation between the hypo-
dermal and cone nuclei. Neither does the cone mass show the
boundaries of its component cells. In stages a little later the cone
cells begin to show individual outlines and the cytoplasm appears
more condensed. Cone formation is practically complete, how-
ever, before the corneal hypodermal and cone. cells show a distinct
line of separation (Fig. 66).

As the cone cells differentiate the cytoplasm becomes less and
less vacuolate and gradually assumes a dense granular appear-
ance. ‘The cytoplasm is most condensed just below the nuclei and
decreases in density proximally.

In longitudinal sections of the cones the cell boundaries appear
distinct from their outer ends inward to the outer retinule. At
this point the cell boundaries become indistinct and the cone
tapers rather suddenly into a slender stalk which extends to the
distal end of the rhabdom, where it ends abruptly (Fig. 66).
At a somewhat more advanced stage the boundaries between the
cone and corneal hypodermal cells become distinct, and the cone
secretion takes on the dense homogeneous deeply staining appear-
ance characteristic of that in mature cones (Fig. 67).

194 M ary Isabelle Steele
c Regeneration of the Rhabdoms

There is no indication that the retinular cells have begun to
secrete the rhabdoms until after the cones have been distinctly
outlined, although the retinula themselves become clearly differen-
tiated before there is any indication of the cones. Not until the
differentiation of the ommatidia has reached a stage intermediate
between these stages shown in Figs. 64 and 66 can the anlagen of
the rhabdoms be recognized. The rhabdoms first appear as
slender homogeneous rods. Each rod is of uniform diameter
throughout its length, and is distinguishable from the inner ends of
the cone cells only by the fact that it stains slightly deeper and
shows no divisions which indicate that it is composed of more than
one cell (Fig. 66.) The rhabdoms show no signs of the character-
istic spindle-like form and the complicated system of transverse
plates so noticeable in the normal adult eye until after the last
stage in the differentiation of the cones (Fig. 67). Even at the
stage shown in the preceding figure the rhabdom does not show a
normal appearance of its spindle form and the pigmented exten-
sions of the retinular do not cover it so completely as in normal
adult ommatidia.

It is evident from the preceding description and accompanying
figures that all the structures necessary to a completely regenerated
eye have been laid down. It is also seen that with the exception
of the corneal facets the regenerated ommatidia are practically
identical with those of the normal adult eye. A specimen of later
stage, however, shows both the corneal facets and the definitive
form of the rhabdom, so that the regenerated ommatidia present a
perfectly normal appearance even to the minutest detail (Fig. 76).

All observations show that the differentiation of corneal facets
could not become evident until after at least two moults. The
first cuticle which is developed is formed before a continuous
hypodermis has grown over the wounded surface and before any
regeneration of ommatidia has begun. Corneal hypodermal cells
are not differentiated as such until all of the other ommatidial
structures have been laid down. ‘Therefore the secretion of
corneal facets constitutes the final process in the regeneration of

Regeneration In Compound Eyes of Crustacea 195

ommatidia. Since this is true, several moults may occur before
the corneal facets differentiate, and at least two must take place.

3 Comparison of Ommatidia in Regeneration and Ontogeny

In comparing the regenerating with the embryonic eye it 1s
necessary to consider them only from the beginning of ommatidial
differentiation, since there can be no exact parallel between the
preliminary stages of regeneration and the mode of the origin of
the embryonic eye. These two processes are similar, however, in
the respect that the ommatidia in both cases develop from the
hypodermal cells. All these observations upon the regenerating
eye give evidence that the cells which take part in the formation
of the new ommatidia are derived primarily from the hypodermal
cells that cover the wounded surface. That the ommatidia of the
embryonic eye develop entirely from the hypodermis is the con-
clusion of most observers. ‘There is no further agreement, how-
ever, in details except in the case of embryonic eyes that are
described as arising without invagination, e. g., the compound
eye of the honey bee as described by Phillips (05) and of the
lobster (Parker ’g0), in which it was found that the ommatidia are
developed from a single epithelial layer and consequently from
morphologically similar cells.

It has been seen that the first cells differentiated from the hypo-
dermis in the regenerating eye are the retinular cells. “This can be
regarded as being in agreement with the conclusion of Phillips
that the retinulz constitute the morphological center of the
ommatidium. At any rate the retinula are in each case differen-
tiated before the cone cells can be recognized, none of the cells
originally separated from the hypodermis to form the retinulz
ever take any part in the formation of cones, and finally the cone
cells arise peripheral to the retinule.

The differentiation of the regenerating ommatidia, described in
a preceding section, and of the embryonic eye, as described by
Kingsley (’87), may perhaps be regarded as presenting a parallel.
Kingsley finds the nuclei which go to make up the cones and re-
tinulz arranged in radial rows and that the outer and hence the

196 Mary Isa belle Steele

later formed nucleus of each row contributes to the formation of a
cone while the remaining nuclei form the retinule.

Unlike the rows of nuclei described by Kingsley and the spindle
shaped groups of cells described by Phillips the retinulz of the
regenerating eye do not appear to become separated into definite
groups before the development of the cones. In sections from
the same eye there may be groups of retinula somewhat distinctly
separated from each other and other retinula which constitute a
continuous band for a considerable distance. Figs. 60 and 61
illustrate these opposite cases. But even when groups can be
recognized there is no certain indication that a group belongs to a
single ommatidium. The group may contain a fewer or a greater
number of cells than belong to a single ommatidium. Besides
the retinule continue to divide occasionally up to the time the
cones are differentiated. From the evidence furnished by a num-
ber of different specimens it appears that the definite separation
of the retinul into groups does not take place until after the cones
are well advanced in their development. As the cones differen-
tiate from the periphery inward the retinulae become grouped
about them. As this grouping continues the retinular processes
become more and more slender, perhaps largely as a result of
mechanical pressure.

The development of the cone as shown by my observations is the
result of intra-cellular secretion. In this respect it agrees with the
embiyonic development of the cones in the eye of the honey bee
as described by Phillips. It is directly opposed to the method
described by Patten (87), Kingsley (’87) and Watase (’89), who
regard the cones as the result of the extra-cellular secretion of the
cone cells.

The evidence furnished by the regenerating eyes of Palamonetes,
Crangon and hermit crabs agrees with the observations of those
who do not find the cone and the rhabdom to be developed i in the
embryonic eye as continuous structures. Some investigators
regard the rhabdom as merely an inward prolongation of the cone
cells. Kingsley finds such a relationship in the embryonic develop-
ment of the eye of Crangon. Patten regards the cone as extending
from the hypodermis to the basement membrane and as differen-

Regeneration in Compound Eyes of Crustacea 197

tiating at the lower end into the rhabdom in most cases. But in
Vespa he describes the inward prolongation of the cone cells as
enclosing the rhabdom. Parker (’00) describes the prolongation
of the cone cells in Homarus as extending to the basement mem-
brane and inclosing the rhabdom in the same manner. ‘The
description of the relation of the rhabdom to the cone in Crangon,
given by Kingsley, and applied to Crustacea in general by Patten,
does not agree with the facts presented by the regenerating eyes of
Crangon, Palzmonetes and hermit crabs. Obviously, however,
this interpretation is in accord with that of Phillips in the case of
the honey bee and that of Grenacher (’74), both of whom find
that the rhabdom is developed as a secretion of the retinulz, and
do not find the cone cells extending as slender processes beyond
the distal end of the rhabdom.

Concerning the source and manner of the innervation of the
ommatidia the results obtained in this study of the regenerated
eye agree only with those observers who, like Parker (’g1) and
Phillips (05), regard the retinular cells as hypodermal sense cells
which send nerve fibers into the ganglion below. It is true that no
special nerve methods were used in this work upon the regenerating
eye. But in some specimens at least the prolongation of the retinu-
lar processes into fibers which penetrate the optic ganglion is clearly
evident (Fig. 62). In many other cases the processes can be
traced from the retinular nuclei to the basement membrane and
similar processes are found branching among the ganglion cells
below it. But in no case is there the slightest evidence that the
ganglion cells are sending fibers upward to the regenerating
ommatidia. Consequently there seems to be no room for reason-
able doubt that the retinulz form the nerve endings of the omma-
tidia.

In this particular these results differ from those of Patten,
Kingsley and other workers on the embryological development of
the eye of certain Decapods. ‘These investigators regard the nerve
connections as being formed by the extension of processes upward
from the ganglion cells, through the rhabdom and into the cone.

The observations made in this work seem neither to uphold nor
to oppose the views of those who find that the ganglion cells send

198 Mary Isabelle Steele

processes upward into the retinula during the embryonic devel-
opment. For it is not inconceivable that the innervation of the
ommatidia of a normal eye should be accomplished by the upward
growth of processes from the ganglion cells to the retinulz and
that in the regenerating eye it should be accomplished by proc-
esses growing inward from the retinulz to the ganglion cells.
That this is not impossible is suggested by the fact that in regener-
ation tissues are sometimes developed from the same germ layer
while they arise from different germ layers in embryonic develop-
ment.

Several instances are known where muscles in regenerated
appendages arise from the hypodermis although normally they
are of mesodermal origin. Miss Reed (’04) finds this to be true in
the regenerating leg of the crayfish. Ost (’07) notes the same
phenomenon in the regenerating antennz of Oniscus.

It is recognized, then, that certain tissues originating normally
from different germ layers may arise in a regenerating organ from
the same germ layer. It would be at least possible that, although
the nerve connections between the optic ganglion and the retinulz
arise as processes from the ganglion in the development of the
normal eye, they might arise as processes from the retinulz in the
regenerating eye. As even in this case they would develop from
the same germ layer although from different parts of it.

The possibility that the nerve connections may have arisen
differently in the embryonic eye and in the regenerating eye is
conceivable. Yet it seems that the evidence obtained from a
comparative study of the normal adult eye and the regenerating
eye suggests that the nerve processes develop from the retinula
in the normal eye just as in the regenerating eye.

The preceding pages show hae the dev iogaede of the regener-
ating compound eye corresponds in a general way with the embry-
onic development of the compound eye. ‘They also show that the
observations made upon the regenerating eye do not agree entirely
with those of any one worker upon the embryonic development of
the compound eye of Arthropods. In many respects, however,
there is a close similarity between the development of the regener-
ating eyes of Palamonetes and hermit crabs and the process of

Regeneration in Compound Eyes of Crustacea 199

differentiation in the embryonic eye of the lobster as described by
Parker (Joc. cit.) Further, these observations upon regenerating
eyes agree with those of Phillips upon the developing compound
eye of the honey bee in regard to the order of appearance of the
retinulz and cones, in the method of innervation of the ommatidia
and in regard to the relation of the cones and rhabdoms. In the
developing eye of the honey bee, however, Phillips finds the rhab-
doms partially differentiated before there is any indication of the
cones. On the other hand in the regenerating eyes of hermit
crabs and Palamonetes the cones are definitely formed before any
thabdoms can be recognized. The variations, however, which
have been noted between the developing compound eye of the
honey bee and the regenerating eyes of Palamonetes and hermit
crabs, cannot be regarded as fundamental. Such differences are
perhaps not more marked than those that would be noted if the
embryonic development of the same eyes were compared.

It is scarcely necessary to add that these observations on regen-
erating eyes are in several respects quite at variance with the
observations of Kingsley and Patten, who find the rhabdoms
developed from an inward prolongation of the cone cells, the cones
formed as extracellular secretions and the ommatidia innervated
by nerve processes coming from the optic ganglion and penetrating
the rhabdoms and cones.

4 Differences in the Regeneration of the Eye Among Pala-

monetes, Crangon and Hermit Crabs

Reference has already been made to the fact that certain differ-
ences in the regenerating eye appear among Palaemonetes, Cr angon
and hermit crabs. The rate of regeneration and the ability to
regenerate varies greatly in these different genera although in the
most essential particulars the regeneration of the ommatidia is
similar. It has been seen that hermit crabs may regenerate an
eye after the removal of half the optic ganglion. But neither
Palamonetes nor Crangon regenerate a perfect eye if the injury
includes any part of the optic ganglion. It has also been seen that
the differentiation of the ommatidia takes place more rapidly in

200 Mary Isabelle Steele

the hermit crabs than in either of the other forms. The only
structure in the eye of the hermit crab which apparently does not
regenerate perfectly is the basement membrane. ‘This membrane,
however, is but slightly developed in the normal eye. It is not
strange, therefore, that it appears imperfect in the regenerated eye.

The regenerated eyes of the hermit crabs in these experiments
have developed from a level below the basement membrane. In
every case they present a clearer and more normal appearance than
the regenerated eye of either Palemonetes or Crangon. This is
largely due to the fact that in the eye of the hermit crab there are
no shapeless masses of old pigment scattered among the regener-
ated tissues as is usually the case in Crangon and Palemonetes.
The absence of the yellow accessory pigment cells in the eye of the
hermit crab also tends to give to the ommatidia a distinct and
orderly arrangement. ‘The absence of this accessory pigment is,
however, not due to incomplete regeneration. “The normal eye of
the hermit crab, unlike that of many Decapods, contains no
accessory pigment cells. These pigment cells are very abundant
in the eyes of Crangon and Palamonetes and tend to make the
ommatidia less clear.

The most significant difference between Palamonetes and
Crangon seems to be in the rate of regeneration after similar
injury. External appearances would indicate that Crangon
regenerates more rapidly than Palamonetes. But a comparative
study of the section shows that the reverse is true.

In almost every individual in the series of twenty Crangon
operated upon the injury was slight. “The wound healed rapidly,
the animals moulted frequently and externally there was every
indication that regeneration was rapidly taking place. An exam-
ination of the sections, however, shows that in none of them has
there been any considerable regeneration. On the other hand,
in each case much of the old injured tissue remained in a semi-
broken down condition.

Sections of the eye represented in Fig. 8 show that one part of the
eye had not been injured below the level of the outer retinular
cells. ‘The proximal ends of the retinule still remain intact
although thirty-one days have elapsed since the operation. A

Regeneration in Compound Eyes of Crustacea 201

continuous hypodermis has not yet been formed. A considerable
area between the cuticle and the outer retinule is occupied by a
granular structureless mass. Just to one side of this area is a
region in which none of the old ommatidia appear but in which
there are new ommatidia almost completely formed. No more
than five of these appear in any one section. ‘These lie near the
basement membrane toward the inner edge of the eye. Con-
sequently these new ommatidia lie next the growing zone, always
present in the eyes of young individuals as described in Section III.
It is apparent then in this particular case that it is impossible to
determine conclusively whether the new ommatidia are regenerated
or normally developed ommatidia. In other cases, however, new
ommatidia are found developing in positions where it is evident
they are regenerating ones.

From the evidence obtained by an examination of a number of
regenerating eyes of Crangon it seems that the rate of regeneration
depends largely upon the rate of removal of the injured tissue.
The failure of the old tissue to degenerate prevents the regeneration
of new structures. Since the injured ommatidia, although they
fail to break down for a considerable time at least, are incapable
of regeneration in themselves. We should perhaps expect the
cones to be incapable of any sort of regeneration for the cone
nuclei have been destroyed and the constructive metabolic activity
of a cell apparently depends largely upon the nucleus. ‘The nuclei
of the retinula, however, have not been injured and still retain
much of their normal appearance. But there is no evidence that
the retinulz ever take part in normal regeneration.

A comparative study of the regenerating eyes of Palaemonetes
and Crangon shows that the difference in the rate of regeneration
is in reality largely a difference in the rate of degeneration of the
injured tissues. In Palamonetes the injured tissues usually
break down rapidly and are quickly removed. In Crangon they
persist indefinitely. Hence regeneration in Palamonetes begins
soon after the injury, and new ommatidia may be almost fully
developed in shorter time than is required by Crangon for the
removal of the injured ommatidia. The specific case of Crangon
perhaps suggests that if all of the ommatidia had been completely

202 Mary Isabelle Steele

removed by the operation, that regeneration would have followed
more rapidly. An inference which is supported by the observa-
tions of Zeleny ('05), who finds that in regenerating appendages of
crayfish, an increase of the injury, increases the rate of regener-
ation. On the other hand, the same inference cannot be applied
to the regenerating eyes of Palamonetes; for as has already been
pointed out for this form new ommatidia differentiate more
rapidly when a part of the old ommatidia remain uninjured.

5 Comparison of Normal and Regenerated Eyes

It seldom happens that a regenerated eye appears altogether
normal either from external or internal examination. Externally
they frequently appear abnormal in shape and are always smaller
than the opposite eye. These external abnormalities are, however,
of no especial importance except in so far as they may indicate
internal conditions. A common external feature which is sugges-
tive of internal conditions is the irregular arrangement of the pig-
ment masses. Internal examination shows that these masses are
frequently remains of broken down retinulz.

Besides the pigment remains of the old retinula, a number of
other abnormalities may appear which make it difficult to interpret
sections correctly. The arrangement of the retinule makes it
difficult to group them into distal and proximal rows of nuclei as
can be done readily in the normal eye. It is quite possible in cross
sections to select ommatiia in which eight retinulz, the typical
number, can be counted but in other cases this number cannot be
recognized owing possibly to the suppression of the eighth retinular
cell, nehicha is eidimeneauy 3 in the normal eye. The difference in
the length of normal and regenerated ommatidia 1s quite noticeable
in many cases. The regenerated ommatidia are often much
shorter. The new ommatidia might have grown to normal size,
however, had the experiment covered a longer period of time.

The remaining significant difference between the normal and
regenerated eyes is that in some regenerated eyes the optic ganglion
is not complete. This difference appears only in the hermit crabs
as these forms may develop an eye after half the optic ganglion has

Regeneration in Compound Eyes of Crustacea 2.03

been removed. In no case is there any evidence that the optic
ganglion regenerates. Consequently this difference would remain
unchanged.

B CASES OF ABERRANT REGENERATION OF OMMATIDIA

In study of the regenerating eye several cases of aberrant regen-
eration of ommatidia have come under observation. One case
deserves especial mention. Fig. 36, a and b represent ventral sur-
face views of an injured right eye and the normal left eye of a
Palemonetes. Judging both from the surface indications and an
examination of the sections the right eye must have been cut off
at a level corresponding approximately with the line a—b shown
in the figure of the normal eye. A cut at this level would remove
the upper two sections of the optic ganglion and injure the third.
_It would also cut across the heavy muscle band lying in the
posterior part of the eye stalk.

The experiment covered thirty days, one moult taking place
ten days after the operation. Casual surface examination was
sufficient to show that a rather large amount of new tissue had
formed and that a spot of pigment had developed on the ventral
side of the stump. The growth of such a large amount of new
tissue is quite unusual when so much of the ganglion has been
removed.

A study of the sections gives additional information regarding
this new tissue. In Fig. 56 the tissue lying between the periphery
and the broken line extending from x to y represents approxi-
mately the amount of new tissue. Careful examination shows
a difference in the character of the differentiacon of the regener-
ated tissues in the different regions. ‘his figure is from a section
so near the dorsal surface that but little of the nerve tissue appears.
Near the right side of the figure a conspicuous section of the old
muscle band is seen. Just distal to the muscle band the new tissue
is more dense and compact than in the remaining part of the
regenerating tissue. “This band of new tissue (mt) is composed of
fibers extending inward from the periphery and joining end to end
with the fibers of the old muscle band, thereby forming a contin-

204 Mary Isabelle Steele

uous band and reéstablishing the broken connection between the
muscle and the chitinous covering of the stalk.

Fig. 57 of the same series represents a section deeper in from
the dorsal surface so that parts of the optic ganglion are apparent.
A few scattered spots of pigment are also present. Here again
the regenerated tissue shows differentiation into strands of fibers in
the part lying beyond the remains of the old muscle band. In
other regions there is a loose network of fibers with scattered nuclei.
A difference, however, in the appearance of the nuclei in different
regions of the regenerated tissue can be observed. From a to b
the nuclei are small and inconspicuous, constituting uniformly
granular masses and staining with but little more intensity than
the fibers which extend inward from them. The nuclei lying
between the points } and c, on the contrary, are conspicuous, stain”
deeply and are more than twice the size of those lying between
aandb. Here also a number of nuclei are seen lying below the
periphery which show a tendency to extend straight inward from
the periphery. Fig. 58 shows the upper part of a section that lies
so near the ventral surface that it is entirely outside most of the
optic ganglion. In this figure no part of the muscle band appears.
Conspicuous masses of pigment are present in this section as well
as a great number of relatively large deeply staining nuclei.
Since the eye was cut longitudinally from the dorsal to the ventral
side the sections near the ventral side are approximately tangential,
so that many of the nuclei that appear to be deep in from the
periphery are in reality near to the surface. ‘This needs to be
kept in mind in interpreting the figures.

It is a conspicuous fact that many of the nuclei shown in Fig. 58
resemble in shape and appearance the retinular nuclei found in
sections of regenerating eyes. Further resemblances between
these and retinular nuclei are their tendency to stain deeply and
the general direction of their long axes which is at right angles to
the surface. These facts considered in relation to each other
leave but little doubt that these elongated nuclei represent the
retinular elements in a regenerating eye. At one point in Fig. 37
(c.c.) the rudiments of two crystalline cones have appeared. This
is additional evidence that an eye is regenerating, imperfect and

Regeneration in Compound Eyes of Crustacea 205
é s

abnormal as it may be. The pigment shown in this figure repre-
sents the maximum amount seen in any section. For the most
part it presents no definite arrangement but lies in irregular
masses clustered within a fairly well defined area. In a few
sections, however, a part of the pigment shows a tendency toward
a normal arrangement as if the pigment granules were contained
within the processes of the retinular cells, and rudimentary
ommatidia can be recognized (Fig. 59).

Any attempt to explain the phenomena presented by the eye
under discussion may appear somewhat premature, since it pre-
sents a practically unique case so that but little data for comparison
is available. In the first place this is the only well established
case of any attempt of Palamonetes to regenerate ommatidial
structures after the removal of a large part of the optic ganglion.
It seldom happens that any regeneration takes place from the eye
stump of Palamonetes when no more than half of the optic gan-
glion remains. Before attempting to explain the phenomena,
therefore, it is well perhaps to examine the results of other observa-
tions that may suggest an explanation.

From the evidence obtained from the study of normally regener-
ating eyes the indications are that the first new regenerated tissue
is largely of an indifferent character, 7. ¢., capable of giving rise to
different structures, as determined by conditions more or less
external to itself. It has been seen that in the regenerating eye
the primary hypodermis gives rise to the cells which develop into
the different structures of the ommatidia. In the earliest begin-
ning of differentiation, if a cell divides so that the plane of division
is at right angles to the surface the two resulting cells are hypo-
dermal cells. If on the other hand the plane of division is parallel
to the surface the inner one of the pair thus formed becomes a
retinular nucleus and the outer one remains hypodermal in char-
acter. At this stage the only apparent difference between the two
nuclei is in their respective positions. In later cell generations
when the division plane is parallel to the surface the inner nuclei
of the pairs formed become crystalline cone nuclei. “Thus we have
cone nuclei, retinular nuclei and hypodermal nuclei indistinguish-
able except for their relative positions. Apparently the subsequent

206 Mary Isabelle Steele

differentiation of each is conditioned by their relative positions in
respect to each other, to the surface and to the old parts present.

That the relation of the old tissues to the regenerating tissues is a
determining factor in the regenerating of the new structures has
been maintained by several workers. Child (?04) who especially
emphasizes the idea, says: “The fate of the new material must be
regarded as depending essentially upon its relation to the old
parts.” That the regeneration of one structure may be dependent
upon the presence of erother has been shown by Lewis (oz). He
found that a lens could be developed from any part of the ectoderm
of a frog embryo by transplanting the optic vesicle and allowing it
to come in contact with the ectoderm. In this case it appears
that the actual contact of the two tissues constituted a determining
factor and that the new conditions have arisen on account of the
new relations of the two tissues.

From the instances referred to the following inferences may be
drawn—first, newly regenerated tissue is largely indifferent in
character; second, the differentiation of the new tissue is largely
conditioned by its relation to the old tissue. “The above inferences
may be used in suggesting an interpretation of the special phe-
nomena under eon nderuian:

To begin, it is evident that the particular individual now being
considered exhibited a more than usual degree of physiological
and regenerative activity. In no other way could we account for
so aici more new tissue than is ordinarily regenerated by a
stump of this length. The sections show that a large part of the
new tissue still presents an undifferentiated appearance, although
in certain regions differentiation has begun. Just above the cut
end of the old muscle band (Fig. 35) the new tissue appears
thickened, arranged in definite fibers and is apparently continuous
with the muscle band suggesting that connections between the
muscle and the chitinous covering of the eye had been reéstab-
lished.

Sections passing through the stump near its ventral side show
rudimentary ommatidia in process of development. Just why
ommatidia should appear on the side of the old stump is not at
first sight apparent. One possible explanation of this phenom-

Regeneration in Compound Eyes of Crustacea 207

enon has, however, suggested itself. The differentiation of new
ommatidia appears to depend largely upon the reéstablishment of
connections between the optic ganglion and the new tissue. ‘That
such is the case is suggested by the fact that in regenerating eyes
the retinular processes reach the optic ganglion before the cones
begin to differentiate and before any pigment is deposited i in the
retinulz. Further, according to Parker (/oc. cit.) inthe embryonic
development of the lobster’s eye from the earliest beginning of
differentiation there is a connection between the ommacaial region
and the optic ganglion and this connection is never lost at any
stage in the development of the eye. Since the pigment appears
in the retinulz after they have formed connection with the ganglion
its presence in this stump (Fig. 36a) may be an indication that the
connections between the optic ganglion and the new tissues have
been formed and consequently that further development of
ommatidial structures has been initiated.

This suggested explanation does not of course give any reason
why ommatidial development should begin on the ventral side
rather than elsewhere. The following Seueiaa.s is suggested.
Cross and longitudinal sections of the roswell eye stump oe that
the optic ganglion extends somewhat nearer the surface toward
the anterior ventral side. On this account perhaps rudimentary
ommatidia have developed on the ventral side first because the
distance between the new tissue and the optic ganglion was shorter
so that nerve connections were more quickly established in that
region than elsewhere.

Other cases of aberrant regeneration have also come under
observation. These, however, have been produced apparently
by pathological conditions. One or two of the more interesting
cases will be described. Fig. 19 represents dorsal surface views
of a Palamonetes eye thirty-one days after the removal of the upper
part of the ommatidial portion. ight days after the operation a
moult occurred. A second moult pecceed fifteen days later and
it was seen that the greater part of the ommatidial structures had
disappeared. Fig. 19 shows a mass of pigment Just distal to the
optic ganglion. Above this pigment there is a considerable area of
transparent tissue which shows no external evidence, however, of

208 Mary Tsabelle Steele

being differentiated into ommatidia. Sections of this eye show a
considerable development of abnormal pigment. At one side are
seen a limited number of regenerating ommatidia that have failed
to differentiate normally. The arrangement of the abnormal pig-
ment is of a character frequently seen in short stumps but not
generally found where the injury, as here, has not involved the
optic ganglion. The probable cause of this abnormal pigment
deposition will be considered in another section. ‘The point of
chief interest here is that we find two sorts of development going
on side by side, one region developing normal structures and a
contiguous region developing abnormal structures.

Another similar, though rather more exaggerated, case is fur-
nished by a small hermit crab (Fig. 25). The eye had been in-
jured before the animal was brought into the laboratory. No moult,
however, had taken place since the injury. About two weeks after
being brought into the laboratory the crab moulted and three days
later was killed. Fig. 25 represents the ventral view both of the
injured and uninjured eye three days after the moult. It is seen
that the injury involved the whole ommatidial portion and appar-
ently a part of the optic ganglion. ‘The distal surface of the
regenerating eye presents a very irregular outline. On the inner
border a peculiar protuberance has developed and two separate
pigment areas are apparent. The upper of these two areas
suggests that small ommatidia have been regenerated. Externally
the lower pigment mass suggests no probable explanation of its
character.

Sections of this eye show a number of interesting points. In the
first place it is demonstrated that the upper and smaller pigment
area belongs to the retinule of small but almost completely
developed ommatidia. All the structures of a typical ommatidium
have been differentiated with the exception of the corneal facets
and the spindle shaped enlargement at the base of the rhabdom.
The lower pigment area is an abnormal pigment deposition similar
to the preceding case.

The protuberance developed on the inner border of the eye
seems also to be formed of abnormal tissue. _ Its interior is entirely
made up of a loose irregular network of tissue containing a number

Regeneration in Compound Eyes of Crustacea 209

of faintly staining nuclei. These tissues resemble very closely the
depigmented tissues of the abnormal pigment masses. ‘This
resemblance suggests that possibly the two abnormal ape sEauee®
have had a common origin.

A case partially resembling the one just described was observed
in a green shrimp, Palemonetes viridis. The original injury
consisted in the removal of a small part of the top of the eye. “The
eye was operated upon August 1. On the ninth of the same month
the animal moulted and was preserved. Figs. 17 and 18 represent
dorsal and ventral views of the injured eye. The dorsal surface of
the whole eye is shown in Fig. 17. Fig. 18 represents the distal
end of the eye from the ene side Ate under greater magnifica-
tion. A pigmented mass similar in general outline to the retinular
area in the normal eye is visible through the transparent outer
tissues. Distal to the pigmented portion is a considerable area of
transparent tissue with flecks of pigment scattered through it.

The distal contour of the eye is irregular because of a swelling or
protuberance similar to the one on the hermit crab’s eye previously
described. This eye also shows an unusual development of new
tissue considering the time in which it was produced.

Sections of this eye show that the optic ganglion had not been
injured, that not all of the ommatidia had been removed and that
a considerable part of the old pigment remained. The ommatidia
that were left have almost completely degenerated, however, and
the whole distal portion of the stump is filled with a complicated
network of faintly staining cells. There is also absolutely no
regularity in cellular arrangement, as is seen in normally regener-
ating eyes. For the most part the nuclei are scarcely distinguish-
able from the cell-body. Although here and there are scattered
nuclei which stain more deeply. There are evidences in some
cases that these are nuclei of disintegrating ommatidial structures.
Some sections show remains of old cones associated with the
darkly staining nuclei. Comparison of others of these deeply
staining nuclei with the nuclei of partially depigmented cells
shows a similarity between the two which suggests that the former
belong to cells in which pigment secretion has lately begun.

It is evident that most of the pigment masses present are the

210 Mary Isabelle Steele

remains of the old ommatidia although they are greatly scattered
through the new tissue. A few dense cysts of new pigment,
however, have been formed and other pigment secreting centers
have begun to appear. From these observations it seems apparent
that had the animal lived the entire mass of tissue sooner or later
would have been densely packed with pigment cysts and that very
probably new eye structures would not have regenerated. For we
have seen in the preceding two cases that an abnormal secretion of
pigment stopped, apparently, ommatidial regeneration after it
had begun. It does not seem too much to assume then that in
this case normal regeneration. of tissues would have been precluded
by such an abundant development of abnormal tissue.

C EYE STUMPS THAT SHOW AN ABNORMAL DEVELOPMENT OR NO
REGENERATION

The instances described in the preceding section apply partic-
ularly to those unusual types in which the ommatidia have begun
to regenerate and this process has been more than balanced by
opposing factors. “This leads naturally to a consideration of cases
in which there is either no regeneration or only an abnormal
development of pigment.

I. Abnormal Development of Pigment

Most of the examples of abnormal pigment secretion were
afforded by Palzmonetes in which the optic ganglion was more or
less injured. Usually in any of the forms studied eye stumps that
contain no more than half of the optic ganglion show no normal
regeneration aside from the cuticle and hypodermis. Any attempt
to regenerate other tissues produces either scattered strands of
connective tissue or abnormal masses of pigment. “These pigment
masses most frequently appear collected in nodules or cysts and
are usually enclosed in a sort of connective tissue sheaths. Fig. 43
represents an eye stump of Palamonetes, showing one of these
pigment depositions. Fig. 72 shows an outline section through
this stump from which the relation of the pigment to the normal
issues can be readily made out. Fig. 43 shows in detail the

Regeneration in Compound Eyes of Crustacea 211

appearance of the pigment outlined in Fig. 72. An examination
of Fig. 73 shows that the deposition of pigment appears to begin
at several centers. These centers gradually increase in size.
There also seems to be a tendency for the several centers to fuse
with each other. It is further seen that the pigment cells or
masses vary from very large to very small areas.

A study of depigmented sections suggests that these smaller
pigment bodies arise in one of two ways: first, by an out-pocketing
of the cytoplasm, which after becoming distended with pigment
separates from the parent mass and second, by an unequal division
of the cell. It is possible that the latter is the true method for all
cases. But it was not possible to determine this point with cer-
tainty. When a pigment cell has become gorged with pigment
the nucleus is much changed and distorted. And even after the
most thorough depigmentation it cannot always be identified.
Consequently it may be that the smaller masses, in which no nuclei
are visible, are not mere masses of cytoplasm that have been
constricted off but are the result of unequal cell division. Fig. 75f
shows a small group of depigmented pigment bodies. In the
larger masses nuclei are visible. In the smaller bodies nuclei
cannot be determined with certainty.

The amount of pigment within a cell varies. Some cells contain
only a few scattered granules while others are so completely filled
that they appear to be black homogeneous masses. In these more
densely filled cells the pigment appears to have fused into solid
brittle masses that can be crushed like starch grains. g in Fig. 75
represents one of these masses after it has been crushed.

The pigment is dissolved from the sections with the greatest
difficulty. Mayer’s chlorine method was generally used for this
purpose. But in removing the cyst-like depositions of pigment it
was found that alternate treatment with the chlorine method and
with one-twentieth per cent KOH in 70 per cent alcohol gave
equally as good and more rapid results. Even with this treatment
twelve to twenty-four hours were required to remove the pigment
from sections 6 thick. Frequently this failed to dissolve the
dense pigment masses. In Fig. 75 the dense, crushed pigment
mass g lies in the same section with the group of depigmented

212 Mary Isabelle Steele

cells shown in f. It frequently happens that not all of the tissues
included in what may be regarded as a single pigment region are
pigmented (Fig. 73). The unpigmented tissues, shown in the
area represented in this figure, contain but few recognizable
nuclei. Here and there are cells that show a few pigment granules
and occasionally small groups of such cells. These facts together
with the general appearance of the tissue suggest that eventually
the entire area might have become packed with pigment.

Figs. 29, 32, 44 represent eye stumps of Palamonetes that show
somewhat different types of these abnormal pigment formations.
Fig. 32 presents a rather unusual type. Externally the pigment
appears as thickly scattered granules instead of a dense black
mass as in most cases. Sections of this stump show a small quan-
tity of new tissue lying at the distal end and alongside the nerve
stump. ‘The cells composing the new tissue are closely packed,
large, granular, and their nuclei do not take up iron haematoxylin
at all. Along the side of the eye stump a number of small pigment
cysts appear but for the most part the cells of the new tissue are
not yet densely pigmented. Many of them, however, show
numerous pigment granules. This particular specimen shows
less of the connective tissue-like, fibrous network than is usually
found in the pigment areas. Apparently this stump shows an
early stage in abnormal pigment secretion. The other two cases
figured show dense masses of pigment. Fig. 44 presents a single
compact mass. In each case sections show the pigment arranged
in the characteristic cysts, such as are seen in Fig. 73.

One additional fact of interest is shown in Fig. 44. The pig-
ment cysts in this case do not lie wholly above and distal to the
remains of the optic ganglion but are embedded in the end of the
optic stump. Apparently the upper part of the ganglion stump
has degenerated and given place to the pigment. ‘This is not an
unique instance as several other stumps have presented a similar
phenomenon. ‘There was one case in particular in which there
were several small pigment cysts embedded in different portions of
the remains of the optic ganglion. ‘The ganglion, in this case had
almost entirely degenerated, apparently. ‘Vhis animal had been
preserved in alcohol, however, and it was consequently impossible

Regeneration in Compound Eyes of Crustacea 213

to determine just how much of the abnormal appearance of the
tissue was due to degeneration before the death of the animal and
how much was due to disintegration after its death.

It is evident that this sort of pigment development, whatever
may be its cause, does not belong to the normal regeneration of an
eye. Further, it appears probable that the causes leading to its
formation are of such a nature that they inhibit the true regenera-
tive process. The last two cases described in the preceding
section furnish evidence of this. In the eyes shown in Figs. 19 and
20 regeneration of normal ommatidia had begun but was limited
by some opposing factor. These causes not only inhibit the true
regenerative processes after they have begun but it is also probably
true that they even prevent true regeneration from beginning.
All the comparative evidence that we have indicates that in the
case of the Palamonetes viridis previously described (Fig. 17) a
new eye would never have developed. The whole distal end of
the stump was filled with a mass of abnormal pigment depositing
cells. Although this case is striking it is not exceptional. Similar
conditions have been found in varying degrees in other eye stumps.
There is sufficient similarity in all the cases of abnormal pigment
deposition to indicate that they have in certain respects a common
cause.

It is important to point out some of these similarities in greater
detail. A striking resemblance exists between the broken down
retinula of an injured eye and the pigment secreting cells. In
the early stages of the disintegration of the ommatidial structures
the nuclei of the retinule frequently become separated from the
retinular processes. Each nucleus becomes surrounded by a
rounded mass of cytoplasm which apparently has no connection
with other structures. ‘The nuclei become polymorphic and not
infrequently appear divided. As the disintegration proceeds these
rounded nuclear cells usually disappear, but, as mentioned in a
preceding section, the broken down masses of pigment remain.
These rounded remains of the retinulz can be identified from a
few hours up to sixteen days after the injury. They are always
seen a few hours after the injury although they may not always be
present in eyes examined in a week to two weeks after the opera-

214 Mary Isabelle Steele

tion. ‘This shows that in some cases they disintegrate much more
rapidly than in others. In some eyes examined twenty-five to
thirty-five days after the operation similar rounded cells with
polymorphic nuclei are found in numbers, increasing by amitotic
division. In still other cases, cells of this character containing
‘pigment granules are found.

Fig. 75a, b, c, d, e, f, g represents a series of groups of these
rounded cells with polymorphic nuclei. ‘These groups were taken
from crayfish, hermit crabs, Crangon and Palamonetes, represen-
ing in all seven species. ‘The first three groups, a, b and c, show
the appearance of breaking down retinula, seventeen hours,
thirty-nine hours and sixteen days, respectively, after the injury;
d, e and 7 show the secretion of abnormal pigment as found in
eyes ten, twenty-three and sixty-seven days respectively after the
injury; 7 represents a group of depigmented cells that were so filled
with pigment that without depigmentation no structures were
visible. An examination of this series cannot fail to show the
similarity between the breaking down retinulz and the pigment
secreting cells. Particularly is this so if it is remembered that,
except a and b, no two groups are taken from the same species.

These facts taken together have suggested that the immediate
cause of the pathological pigment secretion is the abnormal
activity of old retinulz which have not completely broken down.
It has already been mentioned that after an eye has been operated
upon the pigment from the injured retinule frequently becomes
greatly scattered among the other tissues. Not only does the
retinular pigment become scattered but in some cases the rounded
retinular cells, also, are found considerable distances down the
stalk and on the side opposite the injury. ‘These instances were
observed in eyes examined from fifteen to twenty days after the
injury. It seems probable that some of these metamorphosed
retinular cells become embedded in other tissues, then later divide
amitotically and begin to secrete pigment. ‘The nodules of pig-
ment previously described are the result. In some cases the
multiplication of these pathological cells takes place rapidly so
that large areas are occupied by them. Fig. 70, which is from
a section of the eye shown in Fig. 20, represents suchacase. A

Regeneration in Compound Eyes of Crustacea 2155

relatively large amount of new tissue was regenerated by this eye,
and sections show that normal regeneration had begun (Figs. 68,
69). Anew hypodermis was completely differentiated and on one
side the differentiation of ommatidia was taking place (Fig. 69).
The greater part of the new tissue was made up, however, of cells
of a character known to be abnormal. ‘The hypodermal cells are
practically the only cells that appear normal. With the exception
of a small area on one side almost the whole of the interior is filled
with rounded cells containing polymorphic nuclei. Fig. 71
represents a partof Fig. 70 more highly magnified and showing the
structure in greater detail. A comparison of these two figures
with the series shown in Fig. 75 cannot fail to show a striking
similarity.

Altogether there is strong evidence that the failure of the old
retinulz to disintegrate completely is the immediate cause of
abnormal pigment deposition, in many cases at least. Further,
there is some evidence that regenerating retinula may sometimes
become involved in the abnormal secretion of pigment. Group
d, Fig. 75, represents a probable case of this sort. The group was
taken from the regenerating part of the eye in a region where there
is positive evidence that some normal regeneration is taking place.
A few of the cells in the group still show but few pigment granules
and show elongated nuclei, characteristic of regenerating retinulz
(Fig. 75d, ret.n.)

The appearance of the network of tissue with which many of
these pigment nodules are associated still remains to be accounted
for. Evidences which point to the origin of this are not so numer-
ous as are the evidences that the old retinulz form the centers for
the pigment secretion. In some cases these pigment nodules are
found embedded in the hypodermis of the eye stalk, in other cases
in the membrane surrounding the optic ganglion. In such
instances it seems probable that the fibrous network supporting
these nodules is due to the hypertrophy of the normal tissue
immediately surrounding the pigment deposits. In those cases,
however, where a great mass of this fibrous network developed it
seems to have had a different origin. ‘There are three particularly
striking instances of the unusual development of this abnormal

216 Mary Isabelle Steele

tissue, each furnished by a different form. Sections taken from a
crayfish eye fixed sixty-two and a half hours after the operation
show a condition that is apparently an early stage in such develop-
ment. At this stage the network is not yet compact and ts found in
chains of elongated cells, showing nuclei dividing amitotically.
These chains of cells run in all directions but do not appear to
develop from the hypodermis. Some sections show these chains
extending from the injured retinule which still surround the
remains of the old cones (Fig. 74). This suggests that the old
retinular cells are undergoing rapid multiplication.

The chains of cells found in the crayfish eye differ in the follow-
ing respects from the network of abnormal tissue found in Pala-
monetes viridis and hermit crab, shown in Figs. 17 and 25. In
the cases of the hermit crab, Fig. 25, and of Palamonetes viridis,
Fig. 17, the cells constituting the network are no longer recog-
nizable as chains and the nuclei no longer stain deeply nor appear
to be dividing. These differences may be accounted for by the
following facts. First, the whole available space in the stump of
the eye was completely filled with the network in the eyes of hermit
crab and Palemonetes viridis and the chains of cells had become
so completely interwoven that their original character could no
longer be recognized. Second, it is probable that the nuclei no
longer stain deeply because the cells have ceased active division.
It has been shown that the cells cease to divide actively soon after
the secretion of pigment begins. In these cells the secretion of
pigment had begun.

Assuming that these apparent differences have been accounted
for we may now turn to their likenesses which suggest a similarity
of origin. The most suggestive likeness is that in each of the three
forms the abnormal tissue appears to have developed outward
from the base of the wounded area rather than inward from the
periphery. The most striking evidence of this is the fact that
masses of old pigment appear near the periphery as if they had
been carried outward by the growth of the new tissue. These
abnormal tissues, in the case of hermit crabs and Palamonetes, lie
close against the cuticle and several layers of the cells are flattened
as if they were the oldest cells and had been pressed against the

Regeneration in Compound Eyes of Crustacea 217

cuticle by the multiplication of the cells beneath. In these cases
a true hypodermis is not distinguishable. These facts suggest that
the migration and pathological development of the old retinulz
are responsible for most if not all of the cases of abnormal pigment
deposition. This of course does not explain what induces this
pathological development.

The initial cause of this development, in the cases where an
abundant network of tissue has developed, was perhaps due to
some infection at the time of the operation. ‘This is suggested by
the fact that, in the crayfish eye described above, a great deal more
tissue had developed abnormally in sixty-two hours than is usually
developed normally in ten days or two weeks, and by the fact, also,
that in the eye of Palamonetes viridis a very unusual amount of
new tissue had developed during the first nine days after the injury.
The more frequent cases in which the pigment secreting cells
appear as rounded cells, containing polymorphic nuclei similar to
the disintegrating retinular cells, seem to be produced by causes
somewhat different. In some specimens examined some time
after the injury these cells show no signs of rapid multiplication.
It seems probable that these cells are old retinule that have
retained one of their characteristic functions, the secretion of
pigment. Since it is an observed fact that the old retinulz become
metamorphosed and wander to different parts of the stump where
they have been found dividing amitotically.

While the above facts are strongly in favor of the conclusion that
the abnormal pigment-secreting tissue is due to the development
of old retinular cells yet the proof is not absolute. A series of
stages of this development, not more than two days apart, would
have to be examined in order to be certain of the absolute truth
of this tentative conclusion.

2 Eye Stumps that Show No Regeneration

It now remains to consider the other phase of the subject out-
lined in this section; namely, those cases in which there is no
regeneration further than the healing of the stump. A number of
these cases present anomalies in that there is no apparent reason

218 Mary Isabelle Steele

for their failure to regenerate. There were among the hermit
crabs several parallels between those that regenerated an eye and
those that did not, so far as conditions were concerned. In a
series of fourteen hermit crabs that had the ommatidial portion of
the eye removed five regenerated an eye and nine did not. All
were kept as nearly as possible under the same conditions. The
part of the eye removed in the original operation was about the
same for each individual. All were operated upon at the same
time in the same way. Some of those that regenerated an eye and
some that did not moulted upon the same day after the operation.
Consequently the physical condition of these specimens were
apparently similar.

Compare Fig. 13 and Fig. 21. Each of the hermit crabs from
which these figures were taken moulted twelve days after the
operation. The hermit crab from which Fig. 13 was taken was
killed at the end of thirty-eight days and the other at the end of
sixty-seven days. The latter lived nearly twice as long yet it
shows no signs of regeneration. More of the optic ganglion
remains in the stump shown in Fig. 21 than in Fig. 13. Again,
compare Fig. 15 with Fig. 21. The two crabs from which these
figures were taken were operated upon at the same time, moulted
approximately upon the same dates and were killed sixty-seven
days after the operation. ‘The stump shown in Fig. 21 shows no
regeneration while the one shown in Fig. 15 has regenerated an eye
perfect in all of its details.

The number of cases might be multiplied but these given are
sufficient to show the parallels presented by individual cases.
Instances of this sort are confined in great part to hermit crabs.
A number of shrimp, however, failed to regenerate even when the
optic ganglion was not injured. The same is true for Crangon
which in several instances failed to regenerate normally even after
the removal of only a small part of the eye.

In most cases sections of such eyes that did not regenerate show
no recognizable pathological conditions. In the case shown in
Fig. 21, however, there was found what seemed to be the beginning
of pathological pigment development. Externally there were no
signs of pigment formation. ‘The regenerated tissue consisted of a

Regeneration in Compound Eyes of Crustacea 219

heavy cuticle, a hypodermis and some loose strands of tissue
extending from the hypodermis to the distal end of the stump of
the optic ganglion. Grouped at the end of optic ganglion stump
and scattered in the loose tissue above it were a few cells of the
characteristic pigment-secreting type. But none of these cells
had yet become densely filled with pigment (Fig. 75¢). It seems
rather improbable that so little abnormal tissue in which scarcely
any secretion of pigment had taken place could have been the sole
cause in the prevention of normal regeneration. Particularly is
this true when it is remembered that instances have been observed
in which practically complete ommatidia were regenerated in eyes
containing great masses of abnormal pigment (Figs. 19, 25).

VI REGENERATION AFTER REMOVAL OF THE GREATER PART
oR ALL OF THE Optic GANGLION

There now remains for discussion those cases in which the
whole or most of the eye stalk was removed and consequently
either all or the greater part of the optic ganglion. Palamonetes,
Crangon and Heemit crabs will each be considered independently
since Mihie differences presented by them are such as to require
separate treatment.

A HERMIT CRABS

Of a total of sixty hermit crabs operated upon twelve died as a
result of the operation, a loss of 20 per cent as against 55 per cent
of the Palamonetes after a similar operation. ‘Thirty-six of these
remaining crabs moulted from one to three times and lived from
twenty-three to one hundred and ninety-four days. These thirty-
six crabs fall into two groups: those that regenerated an antenna-
like appendage in place of an eye and those that showed no particu-
lar regeneration.

I Regeneration of Heteromorphic A ppendages

Ten crabs in all regenerated an appendage from the old eye
stump. In but one case was more than thirty-two days required
for the appendage to become apparent. All of these appendages
are very small none exceeding in length the normal eye stalk. It

220 Ma ry Isabelle Steele

is probable, however, that they would have increased both in
diameter and length had the experiment covered a longer period
of time. None were distinguishable before the occurrence of a
moult. In each case recorded the appendage appeared after the
first moult. Most of these appendages were definitely segmented
after the first moult, in some instances several segments being
developed within twenty-one or two days. But none show any
indication of being divided into parts corresponding to the exo-
and endopodites.

Figs. 23 and 30 show two appendages that were present twenty-
one and twenty-two days respectively after the injury. Neither
appendage exceeds in length the squame at the base of the normal
eye stalk which measures but little more than one-fourth of the
whole length of the normal stalk. One appendage bears a con-
siderable number of large tubular hairs. ‘The other shows none
whatever. Each is seen to consist of several segments. Five
segments are distinctly visible in Fig. 23 while in Fig. 30 there are
six or seven though they are not distinctly differentiated. In Fig.
30 the appendage projects outward at a broad angle. Fig. 23 1s
unique in that it curves in toward the median line nl suggests in
its general shape and position the squame at the base of the
opposite eye. The bifid tip of this appendage is probably due to
some injury that occurred at the time of the moult. This explan-
ation is suggested by an examination of the specimen.

Figs. 34 and 45 represent two other appendages that appeared
twenty-nine and thirty-two days, respectively, after the injury.
These types differ somewhat from the preceding two. It is to be
noted in both cases that the original operation did not include the
squame at the base of the eye. ‘This is a good indication that at
least a part of the proximal segment of the optic ganglion was left.
The specimen shown in Fig. 34 was sufficiently transparent so that
the optic nerve could be observed extending into the base of the
segmented appendage. ‘The specimen from which Fig. 45 was
taken was fixed in Flemming and the consequent darkening of the
tissues prevented an accurate determination of the length of the
stump of the optic nerve. But the nerve stump could be seen
extending well into the base of the new appendage.

Regeneration in Compound Eyes of Crustacea 221

The appendage shown in Fig. 28 developed in twenty-four days
with the intervention of one moult. It is of interest because of the
indications that the optic nerve has extended through almost the
entire length of the regenerated appendage. It is also of interest
because of the ganglionic swelling that appears to be associated
with the nerve in its distal half.

Fig. 41 shows an unique type in that the appendage is curved
closely back until the free end almost touches the head. Although
this appendage is made up of several segments it was rigid from
its first appearance.

The remaining examples of these appendages are of approxi-
mately the same character as those figured. They belong chiefly
to the type shown in Fig. 34, except that two of them show a
larger number of tubular hairs. One of these belongs to a speci-
men that moulted twice and was not killed for sixty-seven days
after the operation. ‘The regenerated appendage shows but little
advance over those that were fixed at the end of half that time.
It is still no longer than the normal eye stalk and shows no greater
number of sensory hairs than are seen in Fig. 30. ‘The additional
facts obtained from an examination of the sections will be referred
to at the close of this section in the general discussion of their
significance.

2 Cases that Show No Espectal Regeneration

As was stated above out of the thirty-six crabs that moulted one
or more times only ten developed heteromorphic appendages
while twenty-six showed no particular regeneration. ‘The propor-
tion 1s a little more than 30 per cent to a little less than 70 per cent
in favor of those that showed merely a healed over stump.

The stumps that show no actual regeneration present a variety
of shapes and characters. None of them, however, show any
signs of pathological pigment development. From all appear-
ances the failure to regenerate in most instances was due to a lack
of sufficient regenerative activity to produce the new tissue neces-
sary. In some cases where the eye was taken off even with the
head the wound healed over leaving a smooth surface, not so much
as a slight elevation marking the former position of the eye. In

222 Mary Isabelle Steele

most instances, however, a longer or a shorter stump remained.
It is impossible to determine by surface examinations how large
the stump was originally for it decreases in size after the operation.
Sometimes the stump of the optic nerve and ganglion shrinks to
two-thirds of its original volume. Fig. 31 shows a short rounded
stump which evidently contains a part of the proximal segment of
the optic ganglion. ‘The stump, originally as broad as the base of
the opposite eye, has, after one moult twenty-three days after the
removal of the eye, shrunk to one-half of the original mass. The
remains of the optic nerve seem to come flush against the end of
the stump, showing that no new tissue has been developed distal
to it. Fig. 39, thirty-two days after the injury, shows a stump
more than one-third the length of the normal eye. Yet sections
show no indication that any definite structure is being regenerated.
It is useless to multiply figures on this phase of the question. They
only serve to show how completely is lacking any indication of
regeneration.

The following table will serve to show that time cannot be
considered the chief factor in regeneration.

The last five examples given in the preceding table are taken
from the same series. Evidently the conditions here were more
favorable than usual. The original number of the series was
twenty-five. Six of these died either from the effects of the oper-
ation or soon afterwards. Of those remaining five others were
lost through an accident. Out of the fourteen for which there is a

Regeneration in Compound E yes of Crustacea 223

complete record nine developed heteromorphic appendages and all
of them within thirty-three days. Two of the remaining five,
which moulted at the end of twenty-one days and then died, might
perhaps have developed an appendage had they lived through a
second moult. Each of them showed a very small bud where the
eye had been removed.

B CRANGON
I Regeneration of Heteromorphic A ppendages

In some respects Crangon appears to be a favorable form for
experimental work. ‘They are less disastrously affected by the
operation than the others worked upon. ‘The entire eye was
removed from twenty-two Crangon and not one of the number
died from the effects of the operation.

This entire number was of the same series. ‘The experiment
covered a period of thirty-three days, August 3 to September 4,
inclusive. During that time with one exception each individual
moulted at least once and fourteen moulted a second time. ‘Three
of those that moulted but once were eaten by their comrades soon
after the moult. The evident hardiness of the Crangon and the
frequency of the moults would seem to be favorable conditions for
regeneration.

Results, however, show only one individual that regenerated a
heteromorphic appendage, the others showing no regeneration.
Fig. 38a and b shows surface views of this one regenerated ap-
pendage. The animal which developed it moulted on the fourth
day after the operation. At that time there was no evidence of
regeneration. Seventeen days later another moult occurred and
an appendage of six segments, with sensory hairs near the tip,
appeared. The appendage measures four-fifths of the length of
the eye on the opposite side and projects forward at the same
angle. The outline of the optic nerve can be seen extending
through the proximal half of the appendage.

2 Cases that Show No Especial Regeneration

Twenty-one out of twenty-two Crangon showed no regeneration.
Four of these died within nine days after the operation and so

224 Mary Isabelle Steele

perhaps should not be counted either way. ‘There were then
seventeen negative cases against a single positive case.

The eye stalks of Crangon are very short and the sections of the
optic ganglion are crowded very close together, and extend well
into the base of the stalk. Hence it not infrequently happened
that a part of the ganglion remained in the stump. A number of
these stumps have been sectioned and none of them show any
regenerated tissue except the hypodermis and cuticle. Spots of
pigment are often seen at the end of the stump but since the whole
stalk of the normal eye is heavily pigmented this does not seem to
be significant. Figs. 24, 37, 40 and 42 show a variety of appear-
ances which the stumps presented. The accompanying table
shows the number of moults which occurred.

In some cases the eye stump is extremely short while in others
it is longer so that a part of the ganglion remains. All of the
specimens included in the table except No. 17 have been sectioned
but none of them show any signs of regeneration. Sections of No.
10 show that nearly half of the optic ganglion was left but no
regeneration is taking place. A very much folded and wrinkled
cuticle with short hairs projecting from it covers the stump. Even
No. 8 (Fig. 37), short as it appears, is found to contain the proxi-
mal end of the optic ganglion. In this case the stump has merely
healed over but no new tissue has developed. In several other
instances not shown in figures the eye had been totally removed so
that not even a short stump is visible. In most such cases the
cuticle is wrinkled over the spot where the eye had been. The
wrinkles and folds on some of the stumps figured shows the com-
mon tendency. These folds are chiefly due probably to the
shrinking of the inner tissues of the stump.

Regeneration in Compound Eyes of Crustacea 225
C PALAEMONETES

Out of nearly three hundred Palzmonetes not a single individual
regenerated any sort of an appendage when all or nearly all of the
optic stalk was removed. It is true that more than 50 per cent of
them died from the operation or soon after. Often half or two-
thirds of a series died within twenty-five or thirty minutes after the
operation, and in some instances the proportion was still greater.
(See Table 1.) Palamonetes were by far the least resistant of any
of the forms operated upon. There were, however, over sixty
individuals that lived from twenty to one hundred and twenty-
four days and moulted from one to three times.

Considering the results of these experiments it may be said that
Palemonetes vulgaris does not regenerate an antenna-like append-
age in place of an eye. Herbst would, perhaps, insist that these
results were due to a lack of time or to a failure to remove all of
the optic ganglion. ‘This latter objection in many cases could not
be urged. The eye stalk in Palamonetes is long and the optic
nerve extends well into its base. And in these experiments the eye
was so completely removed that not even the vestige of a stump
remained. Consequently there was no possibility of leaving any
part of the optic ganglion. Part of the brain even was removed
with the eye in two series. In regard to the other objection
naturally there is no positive proof that results might not have
been different in a longer period of time. There are strong
reasons, however, for believing that time would have made no
essential difference. Chief among these reasons is the fact that
in the regeneration of any other organ Palemonetes needs but
little more time than the hermit crabs and less time than Crangon.
In three parallel series of experiments upon the regeneration of the
first antenna after its total extirpation it was found that Pale-
monetes regenerates a first antenna as quickly and as perfectly as
either Crangon or hermit crabs. In another parallel series of
experiments upon the regeneration of the second antenna it was
found that Palamonetes regenerates this appendage rather more
rapidly than either hermit crabs or Crangon. Palamonetes may
regenerate a first or second antenna in about thirty days. Neither

226 Mary Isabelle Steele

hermit crabs nor Crangon regenerate these appendages in less time.
In the regeneration of a functional eye it was seen in a previous
section that hermit crabs regenerate rather more rapidly than
Palzmonetes but that Palamonetes regenerate more rapidly than
Crangon. Palamonetes may regenerate a functional eye in thirty
to thirty-five days. It is seen, therefore, that appendages are
regenerated by Palzmonetes in approximately the same time as
they are regenerated in hermit crabs and Crangon. Consequently
it does not seem to be assuming too much to express the conviction
that a greater amount of time would have made no essential
difference in the results of these experiments in which the entire
eye of Palaemonetes was removed.

Below 1s a brief table showing results obtained by removing the
entire eye of Palamonetes. This table does not include all the
individuals of any one series but it is entirely representative.

No. of

spec- | Pexpeximent First moult | Second moult Eaperoen Days esos
nen | begun | closed ation

|
I November 5 | November 27 | December30 | February 3 Cle} none
2 | November g | November January | March 13 124 | none
3 November 9g | November January February 12 95 | mone
4 January 1] January 15 | February 7 February 2 55 | none
5 January 1 | February 20} none | February 28 59 | mone
6 | March 5 | April 24 | none | April 24 50 | none
7 | April 19 | May 5 | June 8&25 | July 3 71 | none
8 May 10 | June none | July 4 55 | none
9 July ~ 10 | July 26 | August 6 | September 2 54 none
10 July 10 | July 27 {August 9\ September 14 66 | none
\ September 6/ |

It will be seen that Palamonetes have been under observation
practically every month in the year. ‘The results in each instance
are negative. Fig. 35a, b, c represent some of the stumps that show
new tissue distal to the nerve stump. Most of the cases, however,

Regeneration mn Compound Eyes of Crustacea 2,2,7

regardless of the time of the experiment and the size of the stump,
are similar to the one shown in Fig. 33, No. 13inthe table. Fig. 33
shows the nerve stump flush against the healed end. ‘The indica-
tions from sections and surface views are not such as to lead one to
expect that further regeneration would have ever taken place.
Fig. 26 represents the only stump that even suggests the develop-
ment of a heteromorphic structure. As the table shows this
specimen lived only twenty-four days, during which time it
moulted twice, and regenerated the tiny mass of new tissue repre-
sented by the darkly stippled portion of the figure. ‘The eye was
completely removed, the cut coming at the level of the attachment
of the eye to the head, represented in Fig. 26 by the line a—b._ Fig.
27 represents a more highly magnified view of the stump.

The remaining figures in the series, Fig. 35a, b,c, show the
maximum regeneration, yet in none of these cases did the experi-
ment cover more than thirty days. Apparently regeneration in
most cases proceeds to the forming of the hypodermis and cuticle,
which may be extended slightly beyond the nerve trunk by loose
strands of connective tissue, and then stops. Fig. 35c shows more
than the usual amount of new tissue. The line a—b represents
the level of the union of the eye with the head. The unshaded
central part of the eye stump shows the remains of the optic nerve;
the shaded peripheral portion shows the new tissue. Neither
sections nor surface examinations give the slightest evidence of the
regeneration of nerve fibers, or of any special differentiation of the
regenerated tissue.

D THE HISTOLOGY OF THE HETEROMORPHIC APPENDAGES

The microscopic structure of the antenna-like appendages has
not been considered in great detail because suitable material has
been wanting. In the whole series of experiments only ten hermit
crabs and one Crangon ever regenerated a heteromorphic append-
age in place of the excised eye. Several of these died and were
preserved in alcohol. From such material no detailed results were
obtainable. Again, the only sections of any particular interest
and value are longitudinal ones. “These heteromorphic append-

228 Mary Isabelle Steele

ages were so small and so curved that it was almost impossible to
obtain satisfactory longitudinal sections.

A few points of interest, however, have been observed. ‘These
for the most part serve to corroborate the observations of Herbst
rather than to add to them. An examination of the appendages
in toto show that the old optic nerve either extended as a nerve
trunk through the greater part of the length of the regenerated
appendage or that other structures were developed in the new
appendage which appeared to be continuous with the old optic
stump (Figs. 30, 34, 38). Sections confirm the observations made
from surface examinations. A large number of intermediate
stages would be necessary, however, to determine whether the
regeneration of the nerve trunk had been from the optic nerve
stump outward or whether peripheral regeneration had developed
nerve fibers inward which unite with the optic nerve stump. ‘The
fact, however, that the nerve trunk appears more distinctly differ-
entiated in the proximal part of the appendage than in the distal
may probably be regarded as an indication that the regeneration
proceeds from the proximal end outward.

Sections of these appendages show that the interior is chiefly
occupied by nerve cells and fibers. “The nerve fibers appear to be
continuous with the nerve fibers of the old optic nerve stump.
The nerve cells are grouped into ganglion-like masses which are
scattered pretty generally through the length of the appendage.
The brain sheath 1s continuous a the loose fibrous sheath which
envelops the mass of nerve cells and fibers.

Fig. 77 shows a somewhat diagrammatic section through the
brain and the proximal end of a heteromorphic appendage that
developed within sixty-seven days after the operation. It was
necessary to combine two sections in order to show the continuity
of the optic nerve with the nerve trunk of the appendage. ‘There
can be no doubt, however, that they form a continuous structure.
One feature is noticeable both in sections and in whole prepara-
tions. That is, that the optic trunk leading to the regenerated
appendage is much smaller in diameter than the one opposite.
This fact suggests that probably only a part of the fibers of the
optic nerve tere persisted (Figs. 34, 38)

Regeneration in Compound Eyes of Crustacea 229

Large blood sinuses are present in the heteromorphic append-
ages. Aside from these no tissues are apparent except the hypo-
dermis and the fibrous sheath which encloses the nerve bundles
and the nerves themselves. In some instances muscles are found
in the base of the appendage but these are probably remains of the
base of the eye stump.

Material has been insufficient to make detailed observations
upon the character of the masses of sensory cells found in the
ganglion-like groups throughout the appendage. Sufhcient obser-
vations have been made, however, to warrant the conclusion that
they are concerned with the innervation of the hollow sensory
hairs. In a few instances processes have been traced into the
bases of the hairs which open by a wide mouth into the interior of
the appendage (Figs. 78 and 79).

Herbst has considered the microscopic structure of these hetero-
morphic appendages in considerable detail and has examined a
number of different stages. He describes the nerve cells as
grouped into spindle-shaped ganglia with groups of nerve fibers
extending from each end of the spindle-shaped masses, the distal
bundle of strands being connected with the sensory hairs while the
proximal bundle passes inward toward the brain. None of the
stages examined by Herbst were younger than about six months,
however, and consequently any structures that had developed
would likely be much more definitely organized than in the
appendages examined in this series of experiments.

Herbst considers that these ganglion-like groups of cells have
developed from the hypodermis and that in the earlier stages they
have no direct connection with the brain. In later stages, how-
ever, he describes the proximal bundles of the several ganglia as
uniting to pass inward to the brain. But in most cases at least he
considers that there is no union with the old optic nerve and con-
sequently that the connection of the appendage with the brain is
secondary. He mentions the similarity between these epithelial
sense cells and those found in the first antenna, homologizing the
sensory hairs which are found on the appendage with the olfactory
sete found upon the first antenna. Finally he comes to the con-
clusion that both in form and structure the heteromorphic append-

230 Mary Isabelle Steele

age shows that it should be regarded as a rudimentary first
antenna. ‘The structure of the heteromorphic appendage regener-
ated by the hermit crab agrees in certain respects with the observa-
tions of Herbst upon the structure of the heteromorphic append-
ages regenerated by other forms. In other respects the obser-
vations made upon hermit crabs are not sufficiently extensive to
have any particular weight either way. The most significant
difference, however, between these observations and Herbst’s is in
regard to the relation of the old optic nerve stump to the nerve
bundles extending through the appendage. In the heteromorphic
appendages regenerated by the hermit crabs there are several
cases in which there can be no doubt as to the continuity of the
optic nerve with the nerves in the appendage (Figs. 28, 34, 38).
Further, these are found in stages younger than any spoken of by
Herbst.

The continuity of the optic nerve stump and the nerve trunk of
the heteromorphic appendage will be considered in all of its
aspects in the general consideration of the problem of such hetero-
morphic regeneration.

E GENERAL CONSIDERATION OF REGENERATION FOLLOWING
REMOVAL OF ENTIRE EYE

It has been seen in some cases that hermit crabs and Crangon
regenerate an antenna-like appendage in place of an eye. On
the other hand, the species of Palamonetes used in this series of
experiments has never shown any indication of such regeneration.
In view of this, the question w hich naturally arises is w hy do we
not find antenna-like appendages growing from the eye stumps of
Palazmonetes vulgaris, when hermit crabs and Crangon kept under
the same condition do regenerate these structures, and when the
phenomenon is of pretty g Sonera occurrence among the Decapods.
Herbst has observed the development of an antenna-like append-
age from the eye stumps of a number of stalked- -eyed Crustacea
belonging to different families. He has even secured a few cases
of this heteromorphosis in another species of Palzmonetes (P.
varians). Morgan (’g9) was the first to make the observation for

Regeneration in Compound Eyes of Crustacea 231

hermit crabs and a like phenomenon has been noted for three
species of crayfish, Cambarus virilis and C. gracilis (Steele ’o4)
and the blind crayfish, C. Pellucidus testi (Zeleny ’06).

Widespread as the phenomenon appears to be, however, no
satisfactory explanation of the cause of such heteromorphic
regeneration has yet been suggested. Also an explanation of the
negative cases, that is, where no particular regeneration takes
place, is equally wanting. In the explanation of any phenomenon
it is essential that negative cases be taken into account before any
general conclusions are drawn. As has been pointed out above,
even among the hermit crabs where the heteromorphic appendages
appeared most frequently, in by far the majority of cases no
regeneration took place. ‘There was in these experiments a single
series of hermit crabs in which nine out of fourteen individuals
regenerated a heteromorphic appendage. In the light of this, we
should perhaps be safe in concluding that for hermit crabs failure
to regenerate may often be due to external conditions. But this
would still explain nothing for Crangon and Palamonetes.

All of the Crangon experimented upon belonged to the same
series and were kept as nearly as possible under precisely the same
conditions. Yet but one out of the original twenty-two developed
an antenna-like appendage notwithstanding there were fourteen
others that lived as long or longer and moulted as frequently. In
so far as it was possible to determine the question, the physiological
activity of the fourteen that showed no regeneration was equal to
that of the one individual that did regenerate the appendage.

Extensive series of Palamonetes were operated upon at the same
time with the hermit crabs, and were kept under similar conditions.
Yet, as has been seen not one regenerated the antenna-like append-
age. From this it appears evident that, whatever variations in
results may be accounted for by differences in external conditions,
the primary answer to the question must be sought elsewhere.

It may be objected that in operating upon the eye the entire
optic ganglion was not always removed. ‘This, however, could
not be offered as an objection 1 in every case. In all three of the
forms there were many instances in which not a vestige of the optic
ganglion remained, and yet no regeneration reamed: Besides

232 Mary Isabelle Steele

there is evidence that in some cases hermit crabs regenerate an
antenna-like appendage when part of the ganglion has been left in
the eye stalk. We have then the following conditions for hermit
crabs at least. First, when the cut comes at a level which leaves
as much as two sections of the optic ganglion intact an eye may
regenerate (a—b, text Fig. 2). Second, when the cut is made at the
base or slightly above the base of the eye stalk (c—d, text Fig. 2) so
that little or none of the optic ganglion remains the regeneration
of an antenna-like appendage is possible. Lastly, if the eye is
removed at a level intermediate between a—b and cd (text Fig. 2)
no regeneration follows.

Text Fig.2 The line a-b represents approximately the level from which a hermit crab may regen-
erate an eye. From the level of the line c-d or below it a heteromorphic appendage may regenerate.
No regeneration takes place from intermediate level, e.g., from the level e-f.

It is possible, perhaps even probable, that the character of the
hypodermis differs more or less at these different levels. It is
even conceivable that the hypodermis should be capable of one
sort of regeneration at the level a—b or above it, and of another
sort at the level c-d or below it; but there is certainly no apparent
reason why no regeneration whatever should take place if the eye
is removed at a plane intermediate between these two levels. So
far as careful microscopic examination can determine there is no
difference in the hypodermal cells underlying the cuticle proximal

Regeneration in Compound Eyes of Crustacea 233

to the basement membrane. Whatever differences in character
may exist between the hypodermal cells over different regions of
the eye, the results of this whole series of experiments suggest the
inference that presence or absence of a maximum amount of the
optic ganglion is a controlling factor in determining the character
of the regeneration. The fact that no regeneration takes place
from levels intermediate between a—b and c-d is in itself evidence
that internal conditions are different at these intermediate levels
than from a higher or a lower level. So far as the optic ganglion
may be a controlling factor the difference in conditions may be due
either to a difference in the character of the ganglion cells or to the
reduced ganglionic mass. From the structure of the optic gan-
glion (Parker ’90 and Kenyon ’g7) it is probable that not until the
lower level c—d has been reached have the peripheral terminations
of the optic nerve fibers been seriously interfered with. Both
Parker and Kenyon mention the fact that a part of the optic nerve
fibers have their cellular origins located in the brain. The fact
that this heteromorphic appendage never regenerates except from
this lower level suggests that there may be a causal connection
between the regeneration of the heteromorphic appendage and the
destruction of the distal terminations of the optic nerve fibers.
With their peripheral terminations destroyed there might probably
be a tendency on the part of the optic nerve fibers to grow outward
and form new terminations. Since their natural terminations, the
cells of the optic ganglion have been destroyed it seems probable
that the fibers of the optic nerve stump would behave like those in a
nerve stump of an ordinary appendage, e.g., a leg or antenna.
This in itself might have a tendency to induce any new tissue that
regenerated to differentiate into the form of some sort of append-
age.

That this heteromorphic appendage should be antenna-like in
form seems probable for two reasons. First, it is the natural
tendency of all Arthropod structures to divide into segments.
Second, the simplest form of joint found in any appendage is in
the antenna. Further, this appendage, although antenna-like,
shows a much greater variety in form than any ordinary regener-
ated appendage and the joints formed are often irregular and

234 Mary Isabelle Steele

incomplete. This fact suggests that the regeneration was not
influenced by a fixed set of internal conditions. In the usual
cases of regeneration and embryonic development, whatever the
determining factor or factors may be, it is recognized that we may
expect certain structures to appear in connection with a given set
of external and internal conditions.

In the development of this heteromorphic appendage, however,
conditions seem more variable. As a consequence it shows con-
siderable variety of form. In some cases the appendage is but
little more than a slender horn-like projection, in other cases the
appendage may be curved inward toward the median line, project
forward at the angle of the eye or curve backward until the free end
touches the margin of the head. (Compare Figs. 23, 38 and 41.)
Again from the very first moult the appendage may appear as a
single flagellum-like structure or as a pair. None of the hermit
babe however, have regenerated a heteromorphic appendage
composed of two flagellum- like parts. But in my_ previous
observations upon cray rash (Joc. cit.) two or three instances were
noted in which the appendage appeared double at the time of the
first moult. Herbst has also noted what he regards as an endo-
podite and exopodite in several instances. ‘The appearance of the
single structure in some cases and the double one in some others
can perhaps be explained by the supposition that the nerve fibers
become separated into two masses in some instances and remain
as a single trunk in others. Miss Reed (/oc. cit.) found that when
the stump of the leg of a crayfish or hermit crab was split longitu-
dinally in some instances two legs were regenerated from a single
stump and in other cases only one. Sections of such legs showed
that the end of the nerve stump had been split in the cases in
which two legs regenerated and that the nerve stump had not been
split when only one leg was regenerated. A similar result might
follow in the development of the heteromorphic appendage if the
nerve trunk became separated into two bundles by the interpo-
sition of another sort of tissue.

An explanation of the antenna-like form of the heteromorphic
appendage having been suggested, attention should now be
directed toward an explanation of its inner structure, which is also

Regeneration in Compound Eyes of Crustacea 235

found to be antenna-like. ‘That its inner structure should be
antenna-like might be expected since its innervation is associated
with a region of the central nervous system that is particularly
concerned with the innervation of the special sense organs, and
since its outward form is antenna-like it is rather to be expected
that the inner structure would also conform more or less to the
antenna type.

It seems evident that the ganglionic groups of sense cells which
are found in the heteromorphic appendage, belong to the general
peripheral nervous system found so widely distributed among the
different Arthropods. The groups of cells and the associated
sensory hairs are equivalent to the “ Hautsinnesorgane”’ of vom
Rath (’94). Ost (loc. cit.), however, does not regard these sense
cells as true ganglion cells, as Herbst does. In the regenerating
antenna of Oniscus, Ost finds the nerve fibers regenerating from
the central stump and the groups of sense cells differentiating from
the hypodermis. ‘The regenerating nerve fibers come from the end
of the nerve stump, extend to the periphery and intermingle with
the sense cells. Bethe (’96) considers that the peripheral nervous
system of Arthropods differs both in function and origin from the
central nervous system. Holmgren (’95) regards it as a sort of
sympathetic system.

That cutting the peripheral terminations of the optic nerve may
induce the regeneration of a heteromorphic appendage seems to
receive some support from the results obtained by Zeleny upon the
blind crayfish. Although reduced in size the optic ganglion is
still present in the rudimentary eyes of blind crayfish. On the
other hand the ommatidial structures are entirely wanting. So
long as the vestigial eye remains undisturbed there seems to be no
tendency toward the development of an antenna-like organ. But
when the optic ganglion is removed a heteromorphic appendage
appears. Such appendages are apparently functional as sense
organs and Zeleny concludes that in the blind crayfish a non-
functional organ has been replaced by a functional one.

The suggested explanation for the outgrowth of the hetero-
morphic appendage also carries with it an implied explanation of
the non-appearance of a heteromorphic structure in place of a

236 Mary Isabelle Steele

somatic appendage. ‘The nerve trunk of an appendage is asso-
ciated with ganglion cells only at its central end, not with ganglion
cells at its peripheral end, as distinguished from the optic nerve in
its relation to the optic ganglion, consequently in removing an
appendage no parts have been removed that would not be likely to
again regenerate in a similar manner. While in animals as highly
specialized as the hermit crabs we do not find the ganglion parts of
the nervous system regenerating.

For the negative cases that appear after the entire optic ganglion
has been removed, it is evident that no real explanation can be
offered until a more adequate understanding of the process of
growth and development has been reached. Although we may
fully recognize the fact that great differences exist in the physiolog-
ical activity of the various individuals and that the external condi-
tions are subject to numerous variations, these facts alone will not
account for the great number of negative cases which result. In
addition to these it seems necessary to recognize an individual
variation in the quality of the tissues. Nothing short of some
specific inherent individual difference seems sufficient account for
the fact that only an occasional hermit crab regenerates a hetero-
morphic appendage. The ability to regenerate a heteromorphic
appendage in place of an eye which appears as an individual

variation in hermit crabs and Crangon and other genera seems to
be entirely wanting in at least one species of Palamonetes. Or if
not entirely wanting it appears so rarely that even after a great
number of experiments and observations it is apparently dbeede

In summing up the foregoing discussion it is apparent that a
weight of responsibility has been placed upon the nervous system.
Numerous observations, however, have left no doubt that the
nervous system does exercise an important physiological influence
upon the other tissues of the body, both in ordinary growth
phenomena and in regeneration. Child (04) observed in oper-
ating upon Leptoplana that if more than half of the cerebral
ganglion was removed a new head did not regenerate. “This was
true regardless of the plane in which the cut was made, a fact
which seems to indicate that the mass of nervous material is an
important factor in the case of Leptoplana at least. Wilson (03)

Regeneration in Compound Eyes of Crustacea 227,

discovered that after the larger chela of Alpheus had been removed,
cutting the nerve in the smaller one prevented it from growing
into the form of the larger one when, however, the large chela had
been removed and the nerve in the small one left intact, the small
chela developed into the form of the large one. It has been noted
above that Miss Reed found she could obtain the regeneration of
the double chelea. The experiments of Schaper (’98), Harrison
(03), Barfurth (or), Goldstein (’04) and others have shown that
the early stages of embryonic development and of regeneration
are apparently independent of the nervous system. But the same
experiments have also shown that the later stages of growth and
differentiation are very largely influenced by the part efihe nervous
system which normally innervates the regenerating or developing
parts; other instances might be mentioned but a sufhcient number
have been given to convince one that in very many instances there
is an important connection between the part of the nervous system
immediately concerned and the regeneration of the other tissues
and structures.

VII REGENERATION AFTER SPLITTING THE Eye Loncirvu-
DINALLY

Several series of Palamonetes were operated upon by having
the eye split longitudinally (Table 1). Although in the regener-
ation of any part of the eye the new tissue is derived from the
hypodermis the results obtained from the experiment of splitting
the eye seem to indicate that injury to the optic ganglion is of
great importance. In many cases at least splitting the eye could
not have resulted in serious injury to the hypodermis yet in-no case
did regeneration follow if the optic ganglion had been injured.
Whether or not regeneration follened the operation apparently
depended upon the depth of the split. If the split extended
through the ommatidial portion only and the optic ganglion
remained uninjured, the ommatidial portion degenerated and new
ommatidia were in some cases regenerated. On the other hand,
if the split extended into the optic ganglion the whole ommatidial
portion and the whole or part of the optic ganglion degenerated.

238 Mary Isabelle Steele

In some cases not even a vestige of the eye remained; in others,
stumps of considerable length persisted. But in no cases where
the split extended into the optic ganglion was there any sign of
regeneration.

Figs. 16 and 22 represent instances in one of which an eye
regenerated and in the other there were no signs of a regenerating
eye. The specimen from which Fig. 16 was taken lived sixty-five
days after the eye was split. The regenerated eye is about six-
sevenths of the length of the normal eye. Sections show that new
ommatidia have regenerated. ‘The eye is not altogether normal
in structure, however. The eye stump shown in Fig. 22 was
taken from an individual that lived seventeen days after the
operation. Apparently the entire optic ganglion has degenerated.
There are no definite indications of regeneration. ‘The stump of
the optic nerve tapers to a point, perhaps indicating that degener-
ation is still incomplete. The stump is little more than one-third
the length of normal eye.

No additional facts of importance were gained from the experi-
ment of splitting the eye. “These results obtained serve chiefly as
additional proof that an injury to any part of the eye 1s followed by
widespread degeneration of the tissues and that in the case of
Palemonetes, after an injury to the optic ganglion usually no
regeneration takes place.

SUMMARY

In summing up the results of the experiments discussed in this
paper the following points are to be noted:

1 The death of the animal which so frequently follows imme-
diately upon the operation is perhaps due rather to its effect upon
the nervous system than to loss of blood.

2 The healing of the wound takes place by the formation of a
provisional crust over the cut surface and later by the development
of a new cuticle beneath this crust.

a The crust is formed of hypodermal cells and a chitinous
secretion. Intermingled with this are blood cells and the cells of
the injured tissues. From two to three days are required for the
formation of the crust.

Regeneration in Compound Eyes of Crustacea 239

b The new cuticle is secreted before a continuous hypodermis
has formed over the wound. It is continuous with the inner
layers of the cuticle over the eye stump.

3 New hypodermal cells over the ommatidial region may arise
in two ways; either by the transformation 7m situ of corneal hypo-
dermal cells into less specialized, actively multiplying hypodermal
cells, or by the proliferation of new hypodermal cells inward from
the edges of the cut.

4 Any injury to the eye is always accompanied by extensive
degeneration of the remaining tissues. Sometimes the entire eye
suffers destruction.

5 The rate of regeneration is considerably affected by the rate
of disintegration and the removal of injured parts.

6 Active regeneration may be in progress at the periphery
while deeper below the surface the injured structures are not yet
removed.

7 In the regeneration of an eye all of the new structures arise
from the hypodermis.

8 Multiplication of cells takes places by amitotic divisions.

9 The cells for the retinule are the first to differentiate from
the hypodermis. ‘Their differentiation may begin before a con-
tinuous hypodermis has developed.

10 The retinular nuclei move inward from the periphery,
elongate and divide along their radial axes, and extend proximal
processes through the basement membranes to the optic ganglion.
Thereby nervous connections are established in the regenerating
region.

1r Not until after the retinular processes have extended into
the optic ganglion is the differentiation of cones established. The
cones differentiate from the periphery inward.

12 The rhabdom is developed from the inner ends of the retinu-
lar cells and is at first present as a slender homogeneous rod of
uniform diameter, which extends from the inner end of the cones
to the basement membrane. The spindle shaped enlargement of
the rhabdom does not appear until after all the other parts of the
ommatidium have been differentiated.

13 The hypodermis does not become a true corneal hypodermis

240 M ary Isabelle Steele

and secrete corneal facets until after all of the other ommatidial
structures have been differentiated. Corneal facets are never
apparent until after more than one moult has taken place.

14 Ommatidia do not differentiate at a uniform rate in all
parts of the regenerating eye.

15 In Palamonetes regeneration of perfect ommatidia does not
take place if the optic ganglion has been injured. -Hermit crabs
may regenerate a perfect eye after removal of as much as half the
optic ganglion. Crangon regenerates an eye very slowly, even
when the optic ganglion is uninjured, but there are evidences that
ommatidia may differentiate after a part of the optic ganglion has
been removed.

16 The rate of regeneration is quite variable in all the species
experimented upon, but both hermit crabs and Palamonetes
however may regenerate ommatidia within thirty-five to forty-five
days.

17 Splitting the eye of Palazmonetes is not followed by regen-
eration if the split extends into the optic ganglion.

18 In the breaking down of the injured ommatidia the pigment
secreting cells become widely scattered, and the old pigment
persists for a long time. Frequent cases of abnormal development
of pigment also occur. “There are evidences which indicate that
this abnormality is due to the pathological development of the
broken down retinulz.

1g After removal of all or nearly all of the optic ganglion,
hermit crabs may regenerate a heteromorphic appendage in place
of the excised eye. There is, however, apparently a level from
which neither an eye nor an antenna-like appendage will regen-
erate.

20 The nerve-trunk of the heteromorphic appendage forms a
continuous structure with the stump of the optic nerve.

21 Removal of the entire eye of Crangon may also be followed
by the regeneration of an antenna-like appendage.

22 In no case was there evidence that Palzmonetes vulgaris
possessed the ability to regenerate a heteromorphic appendage
after the removal of the entire eye.

23 The results of this entire series of experiments points to the

Regeneration in Compound Eyes of Crustacea 241

following conclusion. ‘The regeneration which takes place from
any level is largely influenced by the presence or absence of the
whole or a part of the optic ganglion.

BIBLIOGRAPHY

BarFurtH, D., ’o1—Ist die Regeneration vom Nervensystem abhangig? Verh.
d. Anat. Ges., 1gor (p. 197-201).
BreTtHE, ALBRECHT, ’96—FEin Beitrag zur Kenntnis des peripheren Nervensystems
von Astacus fluviatalis. Anat. Anz., 1896, Bd. xi (p. 31-34).
Cup, C. M., ’04—Amitosis in Moniezia. Anat. Anz., Bd. xxv (p. 545-558).
’o7—Amitosis as a Factor in Normal and Regulatory Growth. Anat.
Anz., Bd. xxx, nos. 11 and 12.
Go.psteEI, K., ’o4—Kritische und experimentelle Beitrage zur Frage nach dem
Einfluss des Centralnervensystem auf die embryonale Entwickelung
und die Regeneration. Arch. f. Entw.-Mech., Bd. xvi, H. 1, 1904.
‘og—Die Abhangigkeit der Muskulatur vom Centralnervensystem wah-
rend derEmbryonalzeit. Eine Erwiderung an Herrn Prof. Neumann.
Arch. f. Entw.-Mech., Bd. xviii, H. 4. :
Grenacuer, H. ’74—Zur Morphologie und Physiologie des facettirten Arthro-
" podenauges. Gdtten Nachrichten (p. 645-656).
Harrison, Ross G., ’03—On the Differentiation of Muscular Tissue when Re-
moved from the Influence of the Nervous System. Am. Jour. of
Anat., vol. 2, no. 2.
Hersst, C., ’96—Ueber die Regeneration von antennenahnlichen Organen an
Stelle von Augen. I. Arch. f. Entw.-Mech., Bd. ii, H. 4.
*g6—Ueber die Regeneration von antennenahnlichen Organen an Stelle
von Augen. II. Versuche mit Sicyonia Sculpta. Vierteljahrsschr.
Naturf. Gesellsch. Ziirich. Jahrg., 41 (p. 435).
*oo—Ueber die Regeneration von antennenahnlichen Organen an Stelle
von Augen. III. Weitere Versuche mit total Exstirpirten Augen.
IV. Versuche mit theilweise abgeschnittenen Augen. Arch, f.
Entw.-Mech., Bd. ix, H. 2 (p. 215-293).
Herrick, F. H., ’89—The Development of the Compound Eye of Alpheus. Zool.
Anz., xii, no. 303 (p. 164-169).
Hormcren, E., ’95—Zur Kenntnis des Hautnervensystems der Arthropoden.
Anat. Anz., Bd. xii, no. 19 (p. 449-457).
Kenyon, F. C.,’g6—The Brain of the Bee. Jr. of Comp. Neur., vol. vi.
‘97—The Optic Lobe of the Bee’s Brain in the Light of Recent Neuro-
logical Methods. Am. Nat., xxxi (p. 369-376).

242 Mary Isabelle Steele

Kincstey, J. S., ’87—The Development of the Compound Eye of Crangon. Jour.
Morph., i, no. 1 (p. 49-66).
McGrecor, J. H., ’99—The Spermatogenesis of Amphiuma. Jour. of Morph.
Sup. to vol. xv.
Meves, g1—Amitotic Division of Spermatagonia of Salamandra. Anat. Anz., Bd.
Vi.
Ost, J., °06—Zur Kenntnis der Regeneration der Extremitaten bei den Arthropo-
den. Arch. f. Entw.-Mech., Bd. xxi, H. 3 (p. 289).
Parker, G. H., ’90—Histology and Development of the Eye in the Lobster. Bull.
Mus. Comp. Zo6l., Harvard, vol. xx, no. 1 (p. 1-60).
*g1—The Compound Eyes in Crustaceans. Bull. Mus. Comp. Zodl.,
Harvard, xxi, no. 2 (p. 45-140).
*95—The Retina and Optic Ganglion in Decapods, Especially in Astacus.
Mitth. Zool. Stat. Neapel., vol. xii, H. 1 (p. 1-73).
*97—Photomechanical Changes in the Retinal Pigment Cells of Palamon-
etes and their Relation to the Central Nervous System. Bull. Mus.
Comp. Zoél., Harvard, xxx (p. 275-300).
Patren, WiiiiaMm, ’86—Eyes of Molluscs and Arthropods, Mitth. Zool. Stat.
Neapel, vol. vi (p. 542-756).
*$87—Studies on the Eyes of Arthropods. 1. Development of the Eyes of
Vespa. Jr. Morph., vol. i (p. 193-226).
Purtuips, E. F., ’05—Structure and Development of the Compound Eye of the
Honey Bee. Proc. Acad. Nat. Sci., Philadelphia (p. 123-157).
Ratu, O. Vom, ’87—Zur Kenntnis der Hautsinnesorgane und des sensiblem Ner-
vensystems bei den Arthropoden. Zeit. f. Wiss. Zool., Bd. 41, H. 2
(p- 499-589).
Reep, Marcaret A., '04—The Regeneration of the First Leg of the Crayfish.
Arch. f. Entw.-Mech., vol. xviii, H. 3 (p. 307-316).
Rosenstapt, B., ‘96—Beitrage zur Kenntnis des Baues der zusammengesetzten
Augen bei den Dekapoden. Arch. Mikr. Anat., Bd. xlvii (p- 748-770).
ScHaPER, A., g8—Experimentelle Studien an Amphibienlarven, Erste Mitteilung.
Haben kinstlich angelegete Defekte des Centralnervensystems oder
die vollstandige Elimination desselben einen nachweisbaren Einfluss
auf die Entwickelung des Gesammtorganismus junger Froschlarven /
Arch, f. Entw.-Mech., Bd. vi (p. 157-197).
*98—Expermental Studies on the Influence of the Central Nervous Sys-
tem upon the Development of the Embryo. Jr. of the Boston Soc.
Med. Sci., January, 18098.
STEELE, Mary I., ’04—Regeneration of Crayfish Appendages. Univ. of Mo.

Studies, vol. 11, no. 4.

Regeneration in Compound Eyes of Crustacea 243

Warase, S., ’89—On the Structure and Development of the Eyes of the Limulus.
Johns Hopkins Univ. Circ., viii, no. 79 (p. 34-37).
*g0—On the Morphology of the Compound Eyes of Arthropods. Studies
Biol. Lab., Johns Hopkins Univ., iv. no. 6 (p. 287-334).
Wison, E. B., ’°03—Notes on the Reversal of Asymmetry in the Regeneration
of the Chele in Alpheus Heterochelis. Biol. Bull., vol. 4 (p. 187-
214).
ZELENY, C., ’05—Compensatory Regulation. Jr. Exp. Zodl., vol. it, no. 1 (pr
102).
’o6—Regeneration of an Antenna-like Organ in Place of the Vestigial
Eye of the Blind Crayfish, C. Pellucidus Testi. Sci., vol. Xxiil (p.
527)-

EXPLANATION OF PLATES

Outlines of all figures were drawn with the aid of a camera. In all of the detailed figures the nuclei
were also drawn in with the camera. The magnification is given with the explanation of each figure.
As far as possible the figures illustrating the different phases of the subject are numbered according to
the number of days the experiment covered.

Reference letters-used

a. anterior. n.ct. new cuticle.
a.cp anterior border of carapace. n.e. normal eye.
a.p.c. abnormal pigment cells. n. nucleus.

bm. basement membrane. nr. nerve trunk.

br. brain. a.ct. old cuticle.
br.sh. brain sheath. cm. old muscle.

c.c. crystalline cones. op.n. optic nerve.
c-hy. corneal hypodermis. 9.sp. optic squame.
c.p. coagulated plasma. p. posterior.

Ch crust. pt. pigment.

ct. cuticle. pt.cs. pigment cysts.
e.s. eye stump. ret, retinula.

cf. corneal facet. ret.n. retinular nuclei.
gl. ganglion. rh. rhabdom.

het. heteromorphic appendage. rt. regenerated tissue.
hy. hypodermis. seg. segments.

hy.tr. transformed hypodermis. s.ct. sub cuticle.

m. muscle. sm. sensory hairs.

Pirate I

Fig. 1 Palemonetes, seven days. One moult seven days after operation. a, Dorsal view, and b,
ventral view. Most of ommatidia removed from ventral side. Pigmented portion appears disorganized.
Injured eye measures about four-fifths length of normal eye. X 45.

Fig. 2 Young Palemonetes, seven days. One moult seven days after operation. Dorsal view.
Eye operated upon by thrusting needle into top of ommatidial portion. Nearly half of the ommatidia
destroyed. Injured eye measures about three-fourths length of normal eye. X 45.

Fig. 3. Palemonetes, ten days. One moult seven days after operation. Ventral view. Part of
ventral ommatidial portion removed. Pigment irregularly scattered throughout ommatidial region.
Very few uninjured ommatidia remains. X 45.

Fig. 4 Palemonetes eye, nineteen days. One moult eighteen days after the operation. Nearly
whole ommatidial portion was removed. Pigment patches remains of old ommatidia. New tissues
can be seen arranged in strands on interior edge. X 45.

Fig. 5 Hermit crab, twenty-five days. Regenerated eye, one moult twenty-four days after opera-
tion. At least one section of optic ganglion removed. Regenerated eye five-eighths length of normal
eye. X 45.

Fig. 6 Palemonetes, thirty days. First moult seven days after operation; second moult twenty-
one days later. Ventral view. Whole ommatidial region destroyed. Upper part of regenerated tissue
perfectly transparent. Irregular patches of old pigment remains seen in lower part of ommatidial
region. Regenerating eye three-fourths length of normaleye. X 45.

Fig.7 Crangon, thirty-two days. First moult eighteen days after operation; second moult fourteen
days later. Dorsal view. Operation removed upper ommatidial surface. Remains of old pigment
apparent. Interior of eye shrunk away from cuticle. Injured eye four-fifths length of normal eye.

X 60.

REGENERATION IN COMPOUND EYES OF CRUSTACEA PLATE I
Mary Isapette STEELE

Tue Journat or ExperiMentar ZobLoGy, VoL. v, No. 2

Pirate II

Fig. 8 Crangon, thirty-one days. One moult sixteen days after_injury. Dorsal view. Operation
removed small part of inner anterior ommatidial surface. Injured eye four-fifths length of normal.
xX 45.

Fig. 9 Palemonetes, thirty-three days. First moult fourteen days after operation; second moult
nine days later. Dorsal view. Injury chiefly on posterior ventral edge. Injured eye four-fifths
length of normal eye. X 35.

Fig. 10 Palemonetes, thirty-three days. First moult fourteen days after operation; second moult
ten days later. Whole of ommatidial region destroyed. Regenerating eye four-fifths length of normal
eye. X 35-

Fig. 11 Palemonetes, thirty-five days. First moult sixteen days after operation. Ventral view.
Part of ommatidia removed from ventral side. Upper end of eye more pointed than usual. Pigment
appears unevenly distributed. Regenerated eye seven-eighths length of normal eye. 45.

Fig. 12 Hermit crab, thirty-three days. First moult thirty-two days after operation. Dorsal view.
Operation removed all of ommatidia and part of optic ganglion. Small, complete, new eye regenerated.
New ommatidia shorter than normal. New eye two-thirds length of normal eye. X 45.

Fig. 13 Hermit crab, thirty-eight days. First moult twelve days after the operation. Dorsal view.
Ommatidial region and nearly half of the ganglion removed. Very small but perfect eye regenerated.
Regenerated eye four-sevenths length of normal eye. X 45.

REGENERATION IN COMPOUND EYES OF CRUSTACEA PLATE II
Mary Isapette STEELE

Tue Journar or ExperiMENTAL ZoLoGy, VOL. v, No. 2

Prate Ll

Fig. 14 Hermit crab, forty-one days. First moult forty-one days after operation. Dorsal view.
Operation destroyed whole ommatidial portion and upper part of ganglion. Complete eye regenerated.
Eye is about two-thirds length of normal eye. X 45.

Fig. 15 Hermit crab, sixty-seven days. One moult forty-five days after operation. Whole omma-
tidial portion and upper part of ganglion destroyed. Regenerated eye fully differentiated. Regener-
ated eye two-thirds length of normaleye. X 45.

Fig. 16 Palemonetes, sixty-five days. Moulted six days after operation. Eye split. New omma-
tidia regenerated. Regenerated eye six-sevenths length of normal eye. 45.

Fig. 17 Palemonetes Viridis, nine days. One moult nine days after operation. Shows irregular
development of upper end of eye. Pigment scattered irregularly. X 45.

Fig. 18 Ventral view of top of eye shown in Fig. 40. Regenerated material appears loose and
reticular. X go.

Fig. 19 Palemonetes, thirty days. Dorsal view. First moult eight days after operation; second
moult fifteen days later. Ommatidial portion wholly destroyed. Upper part of eye transparent. Regen-
erated tissue forms loose reticulum. No external signs of differentiation of ommatidia. Injured eye
seven-eighths length of normal eye. X 45.

REGENERATION IN COMPOUND EYES OF CRUSTACEA PLATE II
Mary Isapette STEELE

Tue Journar or ExperimeNntat ZobLoGy, vou. v, No. 2

Pirate IV

Fig. 20 Palamonetes, thirty-eight days. First moult ten days after operation; second moult four-
teen days later. Ventral view. Entire ommatidial region destroyed. Dark band represents remains
of old pigment. Regenerating eye five-eighths length of normal eye. % 45.

Fig. 21 Hermit crab, sixty-seven days. Moulted twelve days after operation. Stump shows large
part of ganglion remaining but no signs of regenerating eye. Loose shreds of new tissue developed distal
tothe stump. 45.

Fig. 22 Palamonetes, seventeen days. Moulted seventeen days after operation. Eye split. Almost
entire eye degenerated. Stump about one-third length of normal eye. go.

Fig. 23 Hermit crab, twenty-one days. Regenerated heteromorphic appendage and base of nor-
mal eye. One moult twenty-one days after operation. Appendage segmented; curves inward toward
the median line. X go.

Fig. 24 Eye stump, thirty-two days. First moult ten days after operation; second moult sixteen
days later. Pigment patches near distal end of stump. Short stiff hairs on end of stump. Stump is
two-fifths length of normaleye. 45.

Fig.25 Hermitcrab. Injured when found. One moult. Ventral view. Distal end of eye shows
regenerating ommatidia at ». Abnormal pigment developed at pt. Abnormal protuberance on the
inner edge. Injured eye three-fifths length of normal eye. 45.

REGENERATION IN COMPOUND EYES OF CRUSTACEA PLATE IV
Mary IsapeL_e STEELE

Tue JourNat or Exrerimentat ZoOLoGy, vor. v, No. 2

Pirate V

Fig. 26 Palemonetes, twenty-four days. First moult ten days after operation; second moult four-
teen days later. Right eye removed at level of line a-b. Small bud of newtissue. t, Regenerated from
stump. Cuticle removed from stump. % 45.

Fig.27 Stump shown in Fig. 26 more highly magnified. New tissue darkly shaded. X 125.

Fig. 28 Hermit crab, twenty-four days. One moult twenty-two days after removal of eye. Hetero-
morphic appendage. Ventral view. Cuticle heavy, appendage irregularly segmented. Sensory hairs
developing near tip. Nerve trunk visible beyond proximal half of segment. Nerve trunk small in
diameter as compared with Fig. 30. Appendage three-fourths length of normal eye. go.

Fig. 29 Palemonetes, twenty-five days. One moult. Outline of normal eye and eye stump show-
ing abnormal pigment. Pigment in a number of small masses on upper distal end of stump. X 75.

Fig. 30 Hermit crab, twenty-two days. Moulted twenty-two days after operation. Heteromor-
phic appendage. Dorsal view. Show segments, sensory hairs and nerve trunk extending beyond prox-
imal half of appendage. Appendage one-third length of normal eye. 125.

Fig. 31 Hermit crab, twenty-six days. One moult twenty-six days after operation. Dorsal view
of normal eye and healed over stump. Stump three-eighths length of normaleye. X 45.

REGENERATION IN COMPOUND EYES OF CRUSTACEA PLATE V
Mary IsapeLLe STEELE

Tue JourNaL or ExperiMENTAL ZOOLOGY, VOL. V, NO. 2

Prate VI

Fig. 32 Palamonetes, twenty-seven days. First moult sixteen days after operation; second moult
ten days later. Outline of normal eye and eye stump showing abnormal pigment. Pigment appears as
granular area on inner dorsal surface. Eye stump four-fifths length of normaleye. 45.

Fig. 33 Palemonetes, twenty-seven days. First moult ten days after operation; second moult four-
teen days later. Ventral view of stump and normal eye. End of optic nerve stump flush against the
cuticle. Optic nerve reduced in size, two-sevenths lengths of normaleye. X 45.

Fig. 34 Hermit crab, thirty-nine days. Moulted twenty-nine days after operation. Dorsal view
of normal eye and heteromorphic appendage. Shows optic squame in connection with appendage.
Optic nerve stump extends through proximal half of appendage. 35.

Fig. 35 Series of Palemonetes eye stumps after removal of greater part of eye. a, Eye stump with
small quantity of new tissue developed beyond end of optic nerve stump. Stump measures one-third
length of normaleye. X45. b, Eye stump that shows no regeneration. Twenty-ninedays. Moulted
ten days after operation. One-fourth length of normal eye. 45. c, Eye stump showing an un-
usual development of new tissue. Moulted ten days after operation. Stump two-sevenths length of
normal eye. X 45.

REGENERATION IN COMPOUND EYES OF CRUSTACEA PLATE VI

Mary IsaneLte STEELE

Tue Journat or ExrertMeNTAL ZoOLoGY, VOL. v, NO. 2

Prate VII

Fig. 36 a and b, Palemonetes, thirty days. One moult. Optical section of normal and regener-
ating eye. Ventral view. a, Shows regenerating eye; b, normal eye for comparison. Operation
apparently removed eye near level of line a-b on normal eye. Regenerating eye shows considerable new
tissue and pigment spot on ventral side. Heavy cuticle over end of stump. X 45.

Fig. 37 Crangon eye stump, thirty-two days. First moult two days after operation; second moult
nine days later; third moult sixteen days later. Stump one-third length of normal eye. No regenera-
tion. X 45.

Fig. 38 Crangon, thirty-two days. First moult four days after operation; second moult seventeen
days later. a, Optical section of heteromorphic appendage and outline of normal eye. Dorsal view.
45. b, Ventral view of heteromorphic appendage more highly magnified. Shows six segments and
sensory hairs developed on the inner distal edge. Nerve trunk apparent through greater part of length.
Appendage measures four-fifths length of normal eye. go.

Fig. 39 Hermit crab, thirty-two days. One moult thirty-two days after operation. Ventral view
of stump showing no regeneration. Stump two-thirds length of normal eye. X 45.

Fig. 40 Crangon eye stump, thirty-one days. First moult seven days after operation; second moult
twelve days later. Stump measures one-third length of normaleye. X 45.

REGENERATION IN COMPOUND EYES OF CRUSTACEA PLATE VII
Mary IsaneLtte STEELE

Tue JourNnar or ExperiMENTAL ZOOLOGY, VOL. V, NO. 2

Pirate VII

Fig. 41 Hermit crab, thirty-two days. One moult thirty-two days after operation. Dorsal view
of normal eye and heteromorphic appendage. Appendage small and sharply curved backward. Shows
several segments and a few sensory hairs. go.

Fig. 42 Crangon eye stump, thirty-two days. First moult eight days after operation; second moult
twelve days later. Cuticle folded and wrinkled. Short hairs on end of stump. Stump about one-half
length of normal eye. No regeneration. X 45.

Fig. 43 Palemonetes, thirty-eight days. Two moults. Eye stump and outline of normal eye.
Ventral view. Shows abnormal pigment spot. Eye stump one-half length of normal eye. X 45.

Fig. 44 Palamonetes, thirty-eight days. First moult sixteen days after operation; second moult
eighteen days later. Eye stump showing abnormal pigment which appears as a single solid mass on
upper anterior border of eye stump. Stump about two-thirds length of normaleye. X 45.

Fig. 45 Hermit crab, thirty-nine days. One moult. Heteromorphic appendage. Dorsal view.
Nerve trunk distinct in proximal part of appendage. Appendage three-fifths length of normal eye.
X go.

REGENERATION IN COMPOUND EYES OF CRUSTACEA PLATE VIII
Mary Isanette STEELE

Tue Journar or ExreriMENTAL ZOOLOGY, VOL. V, NO. 2

Prate IX

Fig. 46 Semidiagrammatic section through the top of eye of Cambarus virilis sixty-two and one-
half hours after removal of part of ommatidia. Shows relation of cuticle and protective crust (cr.)
Shows broken down tissue excluded by crust also. Below crust is space from which inner tissues have
shrunk. Space occupied by coagulated plasma. go.

Fig. 47 Semidiagrammatic section through upper part of eye of Cambarus gracilis showing con-
tiguity between regenerated and old cuticle. Also shows broken down tissues excluded by development
of cuticle. Eye operated upon by tearing small hole in cornea with needle. 450.

Fig. 48 Section from eye shown in Fig. 9. Broad band of new cuticle developed. Few regenerated
nuclei present. All the tissue shown below cuticle degenerating remains of old ommatidia. 1350.

Fig. 49 Section from eye shown in Fig. 3. Shows new cuticle with no hypodermal cells beneath it.
Shows amitotically dividing nuclei. 1350.

Fig. 50 Part of section from eye shown in Fig. 3. Section from near edge injured area. Hypoder-
mal nuclei much more numerous than in Fig. 51, which is taken from a section near center of injured
area. X 600.

Fig. 51 Section from eye shown in Fig. 3. New cuticle but no well defined hypodermis yet formed
beneath cuticle. Granular masses and pigment patches are remains of degeneratingommatidia. X 430.

REGENERATION IN COMPOUND EYES OF CRUSTACEA PLATE IX
Mary IsapeL__e STEELE

as % 1
48 5 ® 5

Tue Journat or ExperIMENTAL ZOOLOGY, VOL. V, NO. 2

Prate X

Fig. 52 a,b,c, d and e shows series of figures from an eye of Palemonetes representing transforma-
tion of corneal hypodermal cells into active regenerating hypodermis. Shows normal corneal hypoder-
mal cells together with corneal facets and tops of cones ina andb. Transformation of cells from resting
corneal hypodermal cells to active regenerating cells in c and d. These forma continuous series.
X 1350.

Fig. 53 Section of eye of Palemonetes from which part of the ommatidial region was removed.
Shows new cuticle and reticularsubcuticle. Transformed hypodermal cells in process of amitotic divi-
sion. To the left a single cell which may be undergoing mitotic division. Experiment covered twenty-
three days. Moulted twice. Section taken from same eye as series in Fig. 52. X 1350.

Fig. 54 Sections from same eye as Figs. 52 and 53. This section shows cells separating from the
hypodermis and also early stages of differentiation of retinule. Outline of cuticle and subcuticle shown.
X 660.

Fig. 55 Section from eye shown in Fig. 10. Hypodermal cells differentiated. Amitosis taking
place in these and the deeper lying cells. The deeper cells are regenerating retinular cells. 1350.

REGENERATION IN COMPOUND EYES OF CRUSTACEA PUATRE OX
Mary Isapecte STEELE

Tue JourNar or ExreriMENTAL ZOOLOGY, VOL. V, NO. 2

Pirate XI

Figs. 56-59 Taken from sections of regenerating eye shown in Fig. 36. Fig. 56 from section near
dorsal surface. Regenerated tissue lies for the most part peripheral to the broken line xy. Cuticle
torn and inner tissues shrunken from it. Old tissues show parts of muscle bands and small groups of
ganglion cells. Distal to muscle band new tissue seems differentiated into fibers. go.

Fig. 57 Shows section deeper below surface than Fig. 35. Same features as in preceding figure.
In addition a few small pigment masses. An increase in size of the nuclei in region from} toc. Cuticle
not shown. X go.

Fig. 58 Represents upper part of tangental section near the ventral surface. Nuclei increased in
size and number over those in preceding figure. Rudimentary ommatidial elements apparent in new
tissue. Figure composed entirely of regenerated tissue except small group of ganglion cells. X go.

Fig. 59 Rudimentary ommatidia from the eye shown in Fig. 36. Sections oblique so that entire
ommatidium cannot be recognized. Shows distal ends of cones, retinular nuclei and pigmented proc-
esses which appear to be retinule. > goo.

REGENERATION IN COMPOUND EYES OF CRUSTACEA PLATE XI
Mary Isapette STEELE

Tue Journar or ExreriMENTAL ZOOLOGY, VOL. VY, NO. 2

Pirate XII

Fig. 60 Group of retinule from eye shown in Fig. 6. Proximal retinular processes are seen extend-
ing to basement membrane. Two of the processes can be traced through below the basement mem-
brane. X goo.

Fig. 61 Taken from section of eye shown in Fig. 16. Shows group of retinule. Nuclei in out-
line and proximal processes shown. X 1350.

Fig. 62 Group of retinule from eye shown in Fig. 6. Shows proximal retinular processes pene-
trating basement membrane and twining among ganglion cells below. 1350.

Fig. 63 Taken from section of eye shown in Fig. 11. Shows early stage in differentiation of crystal-
line cones. Cone nuclei are being separated from hypodermal nuclei. Hypodermal nuclei are grouped
in pairs. Delicate strands of cytoplasm extending inward from pairs of nuclei. X 1350.

Fig. 64 Taken from section of eye shown in Fig. 11. Shows more advanced stage of cone differen-
tiation than Fig. 29. Cell outlines becoming defined but hypodermal and cone cells not distinctly sepa-
rated. Section somewhat oblique so that the four cone nuclei are visible. Distal retinular processes
extending between the cones. Lower ends of cones not yet differentiated. X goo.

PLATE XII

REGENERATION IN COMPOUND EYES OF CRUSTACEA

Mary Isapeitte STEELE

por,
feet OR agit,

Tue JourNaL or ExreriMENTAL Zo6LoGy, VOL, V, NO. 2

Pirate XII

Fig. 65 Taken from section of eye shown in Fig. 11. Shows retinule with their distal processes
extending to hypodermis. Shows early pigment deposition in proximal processes. Hypodermal cells
shown in outline. XX 1350.

Fig. 66 Taken from section of eye shown in Fig. 11. Ommatidia completely differentiated except
spindle shaped enlargement of the rhabdom. Distal ends of cones not yet differentiated completely.
Cone at left of figure cut obliquely. Retinule not altogether normal in their distribution. X 1350.

Fig. 67 Regenerated ommatidium from eye. Shown in Fig. 11. Rhabdom still not quite normal
in appearance. X 1350.

Fig. 68 Sections from an eye shown in Fig. 15. Shows differentiated hypodermis and retinular
nuclei beginning to assume their definitive position, Hypodermis and retinule both show dividing
nuclei. X 1350.

PLATE XIII

REGENERATION IN COMPOUND EYES OF CRUSTACEA

Mary IsapeLLe STEELE

NO. 2

Tue JourNar or Exrerimentar Zodocy, vor.

Pirate X1V

Fig. 69 Regenerating cones from eye shown in Fig. 20. Most of regenerated part of the eye occu-
pied by abnormal tissue. Abnormal cells mingled with the normally regenerating structures. Com-
pare Figs. 68-71. X 600.

Fig. 70 Part of section of eye shown in Fig. 20. Most of the cells abnormal polymorphic nucleate
cells except those comprising the hypodermis. 450.

Fig. 71 Right hand edge of Fig. 70 more highly magnified. Shows dividing hypodermal cells at
upper edge and cells with polymorphic nuclei in interior. Three retinular nuclei at right edge of figure.
X 1350.

Fig. 72 Outline of section through stump shown in Fig. 43. Shows location of pigment spot with
reference to other structures in stump. X 125.

REGENERATION IN COMPOUND EYES OF CRUSTACEA PLATE XIV
Mary IsaspeL__e STEELE

Tue Journar or Experimenta ZoGLoGy, vot. v, NO. 2

Pirate XV

Fig. 73. Detailed representation of pigment area shown in preceding figure. Shows collection of
pigment cells within cysts. goo.

Fig. 74 Part of section from Cambarus virilis sixty-two and one-half hours after operation. In left
part of figure lower part of cone and upper end of rhabdom. Remainder of figure occupied by chains
of abnormal cells apparently developing from disintegrating retinule. A few cells show pigment gran-
ules. X gIo.

Fig. 75 Group of cells which show polymorphic nuclei. a, Group of disintegrating retinule from
Cambarus virilis seventeen and one-half hours after operation; b, group of disintegrating retinule
from Cambarus virilis thirty-nine hours after operation; c, disintegrating retinule from Cambarus
gracilis sixteen days after operation; d, group of abnormal pigment cells from Crangon twenty-three
days after operation; e, group of abnormal pigment cells from hermit crab sixty-seven days after opera-
tion; f, group of depigmented pigment cells from pigment cyst in eye of stump of Palemonetes thirty
days after operation; g, outline of crushed pigment body lying in same group with depigmented cells
shown inf. X goo.

PLATE XV

REGENERATION IN COMPOUND EYES OF CRUSTACEA

Mary JIsapectte STEELE

Tue JourNAL or ExperIMENTAL ZOOLOGY, VOL. v, NO. 2

Pirate XVI

Fig.76 a, Corneal facet and upper end of cone from fully regenerated ommatidium; }, fully regener-
ated rhabdom a and b both taken from regenerated hermit crab eye shown in Fig. 15. Ommatidia of
hermit crabs much more slender than Palemonetes ommatidia. X goo.

Fig. 77 Hermit crab, sixty-seven days. Section through brain and proximal end of heteromorphic
appendage. Shows continuity of optic nerve and nerve trunk of appendage. Slightly diagrammatic.
xX 1265.

Fig. 78 Section through distal end of heteromorphic appendage, showing strands of fibers sm-f.
extending to the sensory hairs, and groups of sensory cells, s.c. XX 125.

Fig. 79 Detail drawing of small part of section shown in Fig. 78. Shows sensory cells and fibers
sn.f. in connection with bases of two sensory hairs. 750.

REGENERATION IN COMPOUND EYES OF CRUSTACEA PLATE XVI

Mary IsapeL__e STEELE

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Tue Journar or ExperIMENTAL ZOOLOGY, VOL. V, NO, 2

ON SOME PHENOMENA OF COALESCENCE AND
REGENERATION IN SPONGES’

H. V. WILSON

With Four Ficures

In a recent communication I described some degenerative and
regenerative phenomena in sponges and pointed out that a knowl-
edge of these powers made it possible for us to grow sponges in
a new way. ‘The gist of the matter is that silicious sponges when
kept in confinement under proper conditions degenerate in such
a manner that while the bulk of the sponge dies, the cells in certain
regions become aggregated to form lumps of undifferentiated
tissue. Such Taras or plasmodial masses, which may be exceed-
ingly abundant, are often of a rounded shape resembling gem-
mules, more especially the simpler gemmules of marine sponges
(Chalina, e. g.), and were shown to possess in at least one form
(Stylotella) full regenerative power. When isolated they grow
and differentiate producing perfect sponges. I described more-
over a simple method by which plasmodial masses of the same
appearance could be directly produced (in Microciona). The
sponge was kept in aquarium until the degenerative process had
begun. It was then teased with needles so as to liberate cells and
cell agglomerates. ‘These were brought together with the result
that her fused and formed masses sale | in appearance to those
produced in this species when the sponge remains quietly in
aquarium. At the time I was forced to leave it an open question
whether the masses of teased tissue were able to regenerate the
sponge body.

During the past summer’s work at the Beaufort Laboratory?

1 Published with the permission of Hon. Geo. M. Bowers, U. S. Commissioner of Fisheries.
2T am indebted to the director of the station, Mr. H. D. Aller, for his kindly aid in supplying all

facilities needed in the course of my investigation.

Tue Journat or ExreriMENTAL ZOOLOGY, VOL. V, NO. 2.

246 H. V. Wilson

I again took up this question and am now in a position to
state that the dissociated cells of silicious sponges after removal
from the body will combine to form syncytial masses that have
power to differentiate into new sponges. In Microciona, the
form especially worked on, nothing is easier than to obtain by
this method hundreds of young sponges with well developed canal
system and flagellated chambers. How hardy sponges produced
in this artificial way are and how perfectly they will differentiate
the characteristic skeleton, are questions that must be left for
more prolonged experimentation.

Taking up the matter where it had been left at the end of the
preceding summer, | soon found that it was not necessary to allow
the sponge to pass into a degenerative state, but that the fresh and
normal sponge could be used from which to obtain the teased out
cells. Again in order to get the cells in quantity and yet as free
as possible from bits of the parent skeleton, [ devised a substi-
tute for the teasing method. The method adopted is rough but
effective.

Let me briefly describe the facts for Microciona. ‘This species
(M. prolifera Verr.) in the younger state is incrusting. As it
grows older it throws up lobes and this may go so far that the
habitus becomes bushy. The skeletal framework consists of
strong horny fibers with embedded spicules. Lobes of the sponge
are cut into small pieces with scissors and then strained through
fine bolting cloth such as is used for tow nets. A square piece
of cloth is folded like a bag around the bits of sponge and is
immersed in a saucer of filtered sea-water. While the bag is
kept closed with the fingers of one hand it is squeezed between the
arms of a small pair of forceps. The pressure and the elastic
recoil of the skeleton break up the living tissue of the sponge
into its constituent cells, and these pass out through the pores of
the bolting cloth into the surrounding water. The cells, which
pass out in such quantity as to present the appearance of red
clouds, quickly settle down over the bottom of the saucer like a
fine sediment. Enough tissue is squeezed out to cover the bottom
well. The cells display amceboid activities and attach to the
substratum. Moreover they begin at once to fuse with one

Coalescence and Regeneration in S ponges 247

another. After allowing time for the cells to settle and attach,
the water is poured off and fresh sea-water added. ‘The tissue
is freed by currents of the pipette from the bottom and is col-
lected in the center of the saucer. Fusion between the individual
cells has by this time gone on to such an extent that the tissue now
exists in the shape of minute balls or cell conglomerates of a more
or less rounded shape looking to the eye much like small inverte-
brate eggs. Microscopic examination shows that between these
little masses free cells also exist, but the masses are constantly
incorporating such cells. The tissue in this shape is easily
handled. It may be sucked up to fill a pipette and then strewn
over cover glasses, slides, bolting cloth, watch glasses, etc. The
cell conglomerates which are true syncytial masses throw out
pseudopodia all over the surface and neighboring conglomerates
fuse together to form larger masses, some rounded, some irregu-
lar. The details of later behavior vary, being largely dependent
on the amount of tissue which is deposited in a spot, and on the
strength of attachment between the mass of tissue and the sub-
stratum.

Decidedly the best results are obtained when the tissue has been
strewn rather sparsely on slides and covers. The syncytial
masses at first compact and more or less rounded, flatten out
becoming incrusting. They continue to fuse with one another
and thus the whole cover glass may come to be occupied by a
single incrustation, or there may be in the end several such. If
the cover glass is examined at intervals, it will be found that
aii ouertion is gradually taking place. “The dense homogeneous
syncytial mass first dev elops at the surface a thin membrane with
underlying connective tissue (collenchyma). Flagellated cham-
bers make their appearance in great abundance. Canals appear
as isolated spaces which come to connect with one another.
Short oscular tubes with terminal oscula develop as vertical pro-
jections from the flat incrustation. If the incrustation be of any
size it produces several such tubes. The currents from the
oscula are easily observed, and if the cover glass be mounted 1n an
inverted position on a slide the movements of the flagella of the
collar cells may be watched with a high power (Zeiss 2 mm.).

248 H. V. Wilson

This degree of differentiation is attained in the course of six or
seven days when the preparations are kept in laboratory aquaria
(dishes in which the water is changed answer about as well as
running aquaria). Differentiation goes on more rapidly when
the preparation is hung in the open harbor in a live-box (a slide
preparation inclosed in a coarse wire cage is convenient). Sponges
reared in this way have been kept for a couple of weeks. The
currents of water passing through them are certainly active and
the sponges appear to be healthy. In such a sponge spicules are
present, but some of these have unquestionably been carried over
from the parent body along with the squeezed out cells.

The old question of individuality may receive a word here.
Microciona is one of that large class of monaxonid sponges which
lack definite shape and in which the number of oscula is correlated
simply with the size of the mass. While we may look on such a
mass from the phylogenetic standpoint as a corm, we speak of it
as an individual. Yet it is an individual of which with the stroke
of a knife we can make two. Or conversely it is an individual
which may be made to fuse with another, the two forming one.
To such a mass the ordinary idea of the individual is not applic-
able. It is only a mass large or small having the characteristic
organs and tissues of the species but in which the shape of the
whole and the number of the organs are indefinite. As with the
adult so with the lumps of regenerative tissue. They have no
definiteness of shape or size, and their structure is only definite
in so far as the histological character of the syncytial mass is fixed
for the species. A tiny lump may metamorphose into a sponge,
or may first fuse with many such lumps, the aggregate also pro-
ducing but a single sponge although a larger one. In a word we
are not dealing with embryonic bodies of complicated organization
but with a reproductive or regenerative tissue which we may start
on its upward path of differentiation in almost any desired quan-
tity. A striking illustration of this nature of the material is
afforded by the following experiment. ‘The tissue in the shape
of tiny lumps was poured out in such wise that it formed con-
tinuous sheets about one millimeter thick. Such sheets were
then cut into pieces, each about one cubic millimeter. These

Coalescence and Regeneration 1n S ponges 249

were hung in bolting cloth bags in an outside live-box. Some of
the pieces in spite of such rough handling metamorphosed into
functional sponges.

Even where the embryonic bodies of sponges have a fixed
structure and size, as in the case of the ciliated larva, the potential
nature as displayed in later development, is not fixed in the matter
of individuality. Such a body (see p. 10) may form a single
individual or may fuse with some of its fellows to form a larger
individual differing from the one-larva sponge only in size. It
is then in spite of its definiteness of shape and size, essentially
like a lump of regenerative tissue in that whether it develops into
a whole sponge or a part of a sponge depends not on its own
structure but on whether it is given a good opportunity of fusing
with a similar mass. A parallel case to the coalescence of larvae
is afforded by the gemmules of fresh water sponges. Mr. M. E.
Henriksen in a manuscript account submitted to me a year ago,
describes the fusion of gemmules to form a single sponge.

In the preceding description I have passed over the question
as to the precise nature of the cells which combine to form the
masses of regenerative tissue. On this point as on the histological
details in general I hope to have more to say later. Nevertheless
the phenomena are so simple that observation of the living tissue
reveals much, probably indeed all that is of fundamental impor-
tance. If a fairly dense drop of the squeezed out tissue be
mounted at once and examined with a high power (Zeiss 2 mm.,
comp. oc. 6), the preparation is seen to consist of fluid (sea-water)
with a few spicules and myriads of separate cells. The cells
fall into three classes.

1 ‘The most conspicuous and abundant are spheroidal, red-
dish, densely granular, and about (8#/in diameter. These cells
which can be nothing but the unspecialized, amceboid cells of the
mesenchyme (ameebocytes or archzocytes), put out hyaline pseudo-
podia that are sometimes elongated, more often rounded and
blunt.

2 here is also a great abundance of partially transformed
collar cells, each consisting of an elongated body with slender
flagellum. he cell is without a collar, the latter doubtless hay-

250 H. V. Wilson

ing been retracted. In the freshly prepared tissue the flagella
are vibratile, the cells moving about. Soon however the flagellum
ceases to vibrate.

3. The third class is not homogeneous. In it I include more
or less spheroidal cells ranging from the size of the granular cells
down to much smaller ones. Many of these are completely
hyaline, while others consist of hyaline protoplasm containing
one or a few granules.

Fusion of the granular cells begins immediately and in a few
minutes time most of them have united to form small conglomer-
ate masses which at the surface display both blunt and elongated
pseudopodia. ‘These masses soon begin to incorporate the neigh-
boring collar and hyaline cells. One sees collar cells sticking
fast by the end of the long flagellum to the conglomerate mass.
Other collar cells are attached to the mass by short flagella. Still
again only the body of the collar cell projects from the mass while
there is no sign of the flagellum. Similarly spheroidal hyaline
cells of many sizes are found in various stages of fusion with the
granularconglomerate. In sucha preparation the space under the
cover glass is soon occupied by innumerable masses or balls of the
kind just described, between which continue to lie abundant free
cells, some collar cells, others hyaline. Practically all the granu-
lar cells go to make up the balls. The play of pseudopodia at
the periphery of such balls, which results in the incorporation of
free cells and in the fusion of balls to form larger masses, is easily
watched. Along with such a cover glass preparation it is con-
venient to have some of the squeezed-out tissue in a watch glass
of sea-water. In the watch glass preparation it is instructive to
watch with a two-thirds or one-half objective the fusion of the cell
conglomerates to form masses like those strewn on covers, slides,
ete: ((p: 3):

These observations on the early steps in the formation of the
masses of regenerative tissue make it plain that such masses are
composed chiefly of the spheroidal, granular cells (amcebocytes
or archeocytes), but that nevertheless other cells, collar cells and
more or less hyaline cells also enter into their composition. I may
recall the fact that in the formation of regenerative masses in a

Coalescence and Regeneration im Sponges 251

degenerating sponge,? the evidence from sections, which is the
only evidence available in the case, points to the conclusion that
the collar cells help to form the syncytial tissue of the masses.
The question of interest lying at the heart of this matter may be
so formulated: can particles of the Microciona protoplasm dif-
ferentiate into functional collar cells and, when the occasion
arises, change back into unspecialized masses capable of com-
bining with other masses of unspecialized protoplasm to form a
regenerative body? ‘The facts to which I have just alluded sup-
port this idea, and indicate that the immediate problem is one
worth pursuing farther as a good case of temporary differentiation
of protoplasm in the metazoa analogous to the temporary speciali-
zation of the cell individual which occurs in such colonial protozoa
as Protospongia.‘

As far as the amcebocytes are concerned it is certain that
they have great regenerative power. Weltner in a recent paper?
has emphasized the importance of these unspecialized cells in
the processes of growth and regeneration. _ His conclusions which
refer directly to fresh water sponges, are that in a growing
sponge, in a sponge regenerating new organs after its winter
period of simplification, and in the regeneration of a sponge from
a cutting, the amcebocytes are the all-powerful elements in that
they give rise to all the new tissues formed, He further alludes
to the fact that such reproductive bodies as the gemmules of fresh
water sponges and the buds of Tethya (according to Maas) are
only groups of amoebocytes; further that the gemmules of Tedania
and Esperella described by Wilson as developing into ciliated
larve, and the similar bodies found by Ijima in hexactinellids, are
such groups. I may add that the presence of such groups of
unspecialized cells in the hexactinellids has recently been con-
firmed by the master in sponge-morphology, F. E. Schulze, who
recognizes the probability of their reproductive nature and gives

3 A new method by which sponges may be artificially reared, Science, n. s., vol. xxv, no. 649, 1907

* Metschnikoff, Embryologische Studien an Medusen, p. 147, 1886.

5 Spongilliden-studien V. Zur Biologie von Ephydatia fluviatilis und die Bedeutung der Ameebocyten
fiir die Spongilliden. Archiy fiir Naturgeschichte, 73 Jahrg., 1 Bd., 2 Heft, 1907.

252 H. V. Wilson

them a new name, that of sorites.° It is clear then that in many
sponges reproductive bodies are formed by the association of
unspecialized amoeboid cells. But there is nothing in this fact
which precludes the possibility that the groups of amcebocytes
are in part recruited from transformed collar cells and other tissue
cells, such as pinacocytes (flat cells of canal walls), that have
undergone regressive differentiation into an unspecialized amoeboid
condition.

Cells analogous to the amcebocytes of sponges are found else-
where in the metazoa, e. g., in the ascidians.’_ It would be inter-
esting to know what capacity, if any, for development they have,
when freed from the parent (bud) and collected together in sea-
water.

I shall here briefly record some experiments which gave only
negative results but which under circumstances admitting of a
wider choice of species, ought to yield returns of value. These
experiments were based on the assumption that if the dissociated
cells of a species will recombine to form a regenerative mass and
eventually a new sponge, the dissociated cells of two different
species may be made to combine and thus form a composite mass
bearing potentially the two sets of species-characteristics. It is
clear that such an organism would be analogous to one produced
by an association of the blastomeres of the two species. Pending
the successful carrying out of this experiment, it would be idle to
discuss further the nature of the hypothetical dual organism.

In my own experiments three sponges were used: Microciona,
Lissodendoryx and Stylotella. The three are all monactinellids,
but Microciona is the only one in which the skeleton includes any
considerable amount of horny substance. Dissociated cells of
Microciona and Lissodendoryx were mixed, and again dissociated
cells of Microciona were mixed with those of Stylotella. In each
case the experiment was performed at two different times, and a
considerable number of admixtures, in watch glasses and on

6 Wissensch. Ergebn. d. Deutsch. Tiefsee-Exp. 1898-99. | Hexactinellida, pp. 213-15. Jena, 1904.
7 Comp. Hjort’s and Lefevre’s papers on budding in ascidians.

Coalescence and Regeneration in Sponges 253

cover glasses, was made. The preparations were examined at
short intervals with the microscope. The cells of these three
species are colored very differently, and are therefore easily dis-
tinguished, at least as soon as fusion sets in and little masses of
cells begin to be formed. In all the experiments the cells and
cell-masses of a species combined, and not the cells of different *
species. Thus in the admixture of Microciona and Lissoden-
doryx, Microciona regenerative masses and Lissodendoryx regen-
erative masses were produced. Similarly when Microciona and
Stylotella cells were mixed, the resultant masses were pure, some
Microciona, some Stylotella. The Microciona masses in these
experiments were hardy. They continued to develop and in
some preparations metamorphosed. ‘The cell masses of the other
two species while they reached a considerable size were not hardy,
most dying soon although some began the process of metamor-
phosis.

These three species are so unlike that there was little ground in
the beginning for the expectation that coalescence would take
place. Possibly as in the cases where fusion of egg and sperm of
different species is induced through some alteration in the physio-
logical state of the protoplasm, so the regenerative cells and cell
masses of different species may be made to combine under abnor-
mal conditions. The more promising task is however to find
allied species and subspecies, the regenerative tissue of which
will combine under natural conditions. Such forms, I take it,
should be sought among the horny sponges and the monactinellids
with abundant horny matter.

The tendency to fuse so vigorously displayed by the cells and
cell masses of regenerative tissue led me to examine into the
power that larva have to fuse with one another and the capacity
for development in the resultant mass. Delage and others have
remarked on the .ot infrequent occurrence of fusion between
sponge larve. Delage® says that he has often observed two or

8 Embryogénie des Eponges. Arch. de Zool. Exp. et Gén., p. 400, 1892.

254 H. V. Wilson

several larva unite to form a single sponge “which has from the
start several cloacas.”’

I find that this power to fuse displayed by the larvz is one that
is easy to control. Fusion between larve will readily take place
if they are brought in contact at the critical time when the ciliated

Figs. 1, 2,3, 4 Composite masses produced by the fusion of larve. The stippled ends and areas
are in nature blue, and represent the ends of the component larve. The body of the mass is white.
Fig. 1 shows a mass composed of four larva which has just united with a mass composed of five or
six larve. In Fig. 2 more than ten, probably about twenty, larve have combined. In Fig. 3 about
six larve have combined. In Fig. 4 the original quadruple mass composed of four radiately arranged
larva, has been extended in one direction by the addition of a pair of larve, and in the opposite
direction by the addition of two pairs of larve. Figs. 1 and 3 X 44; Figs. 2 and 4 X 22.

epithelium is being replaced by the permanent flat epithelium.
At this time they will fuse in twos or threes or in larger number
up to and over one hundred (Figs. 1-4). The smaller composite
masses composed of as many as five or six larvae metamorphose
into perfect sponges. The larger masses composed of many

Coalescence and Regeneration in Sponges 255

larvae did not metamorphose in my experiments but experience
with the regenerative tissue suggests that such masses would
metamorphose if certain mechanical difficulties due to the great
size of the mass were removed. Possibly this might be accom-
plished by cutting a flattened sheet composed of some hundred
larve (such as I have produced) into pieces and inducing the
pieces to metamorphose separately.

I may now describe some of the details in this process of larva-
fusion. In a species of Lissodendoryx used the larva is of the
following character. It has the usual ovoidal shape with a
posterior protuberant non-ciliated pole. The anterior pole is
somewhat truncated and is sparsely ciliated. The rest of the
body bears the usual thick covering of cilia. As seen with reflected
light the bulk of the body is dead white, the posterior pole deep
blue, and the anterior pole bluish. This coloration is not abso-
lutely fixed for the species, but the larve used in my coalescence
experiments were all of this character. Within twenty-four hours
after liberation the ciliated larve are creeping (remaining in con-
tact with the bottom as they swim) over the bottom of the dish.
Some are now put in deep round watch glasses and with pipette
and needle coaxed together into a clump. Fusion soon begins
and on the next day plenty of composite larva are present. The
larvae fuse endwise, for the most part in pairs. ‘The compound
larva so produced owing to its weight has a very feeble locomotory
power. Using pairs chat are nearly motionless, larvae may be
brought together (coaxed with needle) and arranged in a desired
position on a cover glass for instance. In gna cases fusion
results before the separate masses move apart. In this way,
selecting an instance, | have added to one arm of a quadruple
mass a pair of larva, and to the opposite arm two pairs (Fig. 4).

For the purpose of bringing about the fusion of many larve the
following simple method is convenient. Suppose that we have
the larva in a parafine-coated dish, and they are in a late “creep-
ing” stage. Small excavations, 2-3 mm. deep and 4~5 mm. wide,
are now made in the parafhne, and with the pipette the larvae
are driven into the holes. They lie here in numbers up toand
over one hundred, crowded together and heaped upon one another.

250 H. V. Wilson

Fusion begins soon and the larve are gradually converted into a
flattened cake. The larger cakes thus made measured four by
three millimeters. The body of such a cake is a continuous
flattened mass in which there is no indication of the component
larvae, but the rounded ends of the larvze that have last fused with
the general mass remain for a time distinguishable. Owing to
their blue coloration the ends of the larvae may be recognized in
these and the other compound masses even after the outline of
the larva has been completely lost.

As already stated the smaller compound masses metamorphose
without difficulty. The coalesced larve may be made to attach
to cover glasses, slides, etc. Larger masses composed of about
twenty larvae underwent a partial metamorphosis. Such masses
were laid upon bolting cloth to which they readily attached. The
largest masses were hung in small bolting cloth bags in a live box.
Whether owing to bad handling or more probably to some inherent
difficulty, they did not metamorphose but soon died.

The ease with which larvae of the same species may be made to
fuse together suggests that larvae of different species might like-
wise be induced to coalesce. Some experiments along this line
could not fail to be of interest.

In the tendency to fuse with the production of a plasmodium,
the dissociated cells of sponges resemble the amcebocytes (amce-
bula) of the mycetozoa and Protomyxa. ‘The regenerative power
of the plasmodium has an interest both theoretical and economic
in itself. But it is the tendency to fuse displayed by the cells that
have been forcibly broken apart, which constitutes the fact of
most general physiological importance. Discarding for the mo-
ment the word “cell” and speaking of the protoplasm of a species
as a specific substance, the phenomena may be restated to advan-
tage in the following way.

A mass of sponge protoplasm in the unspecialized state typically
exhibits pseudopodial activities at the surface. In lieu of more
precise knowledge it is useful to regard the pseudopodia as struc-
tures which explore and learn about the environment. Oncoming

EQUILIBRIUM OF ANIMAL FORM?

BY
HANS PRZIBRAM
Biologische Versuchsanstalt, Vienna

Witn Ten Ficures

If the equilibrium of a mass be disturbed, the body will alter
its position in regard to the surrounding neighborhood till it
again gets into a position of equilibrium. If the form of an
animal be altered through amputation of certain parts, the
equilibrium of mass may or may not be altered therewith, the
animal either being able to maintain its position or having to
alter its posture for readjusting its equilibrium. But this is not
all that may happen: it may restore its form, too, after some time,
thus tending toward a new equilibrium of form, till it has reached
a new stable condition.

This may involve three regulatory processes: Regeneration of
lost parts from the cut surface; reduction of existing parts in
contact with the cut surface; compensation at parts of the body
not touched by the amputation.

The study of regeneration has long received much attention;
of late reduction too has been studied more fully, especially in
the lower animals, whilst compensation as a means of restoring
animal form seems first to have been pointed out by me. Its
study has been taken up especially in America, where Zeleny
found additional cases (observed independently of my work),
and Wilson, Morgan and Emmel have studied different aspects
of compensatory regulation in crustaceans.

Having found the principle of compensatory regulation illus-
trated in the chel of Alpheus, I have been looking for other analo-
gous cases and have found the same process in other crustaceans,
especially Callianassa and the common Crabs, Portunus and

1 Read before the International Congress of Zodlogy at Boston on August 22, 1907.

Tue JourNar or ExperIMENTAL ZOOLOGY, VOL. V, No. 2.

260 Hans Prabram

Carcinus, as I demonstrated at the last session of the German
Naturalists’ Association. It will be remembered that this com-
pensatory regulation consists in the hypertypical growth of the
smaller claw of the first pair of thoracic limbs, after autotomic
removal of the big chela, whilst a hypotypical small chela regener-
ates. That such a “transposition” or “reversal” need not be
restricted to this pair of appendages I am now able to show in
Typton spongicola (Fig. 1), where the second pair of thoracic
limbs is developed into asymmetrical chele. After removal of
the bigger chela, which, by the way, may be situated normally at
the wen or at the left side of the body, reversal of the chet of
this second pair is brought about (Fig. 2), the process being in
all respects analogous to that inthe first pair of chelze in Alpheus:

But not only may the means of regeneration and compensation
be clearly shown to occur in this case, but also reduction is
involved to an appreciable degree, especially if the crayfish 1s
starved during the experiment. Then each moult shows the
shedding of a smaller skin and the animal is at the end of the
transposition in all dimensions smaller than at the time of the
operation. Thus, as in the lowest animals, a proportionate
diminution of the whole form may be produced as regulation
proceeds, the only difference with Morgan’s “morphallaxis” lying
in the bigger fragment necessary for reconstruction.

A curious instance of “compensatory reduction” was met in
some experiments on cutting the nerves in crabs. As Morgan
has reported, the chela generally degenerates after this operation.
In a few cases, however, I was fortunate enough to get a further
growth of the limb. In these the terminal joint of the big or
crushing claw was removed (Fig. 3),and in one instance regenerated
in a rather reduced state; but also the corresponding dactylopodite
of the smaller or nipping claw lost its differentiation (Fig. 4).

Compensatory reduction may also occur in animals other than
crustaceans and in other regions than in the chela. Megusar,
working on regeneration in beetles (Coleoptera), amputated one
of the two slightly differing jaws of the Hydrophilus larva. Whilst
there are normally two feeth on the inner side of each jaw (Fig. 5)
the larva appeared after the moult succeeding the amputation

261

Equilibrium of Animal Form

fre. 2.

FLGH:

f16. 4.

HIGHS:

FIG. 6.

Fie. 5.

262 Hans Przbram

of but one jaw with but one tooth on the non-operated jaw, the
regenerating one showing no teeth (Fig. 6).

A tendency ‘toward a quick restoration of a symmetrical con-
dition was also found several times in experiments of Miss Zuel-
zer on the regeneration and moulting periods of the isopod,
Asellus aquaticus. When both long antenne were removed,
simultaneously, but at different levels (Fig. 7) they would be apt
to appear regenerated to an equal length (Fig. 8) even after the
next moult, though not having yet attained their normal length.
In some way these conditions may be related to the different rate
with which appendages regenerate from different levels.

It is not necessary that the two correlated parts be symmet-
rically situated. The male of the water-newt, Triton vulgaris,
produces in its state of courtship a crest along its back and around
its tail, which has a ragged appearance (Fig. 9). Kammerer cut
the tail off to test the regenerative power of the secondary sexual
characters. He found that at first the tail appears with smooth
not ragged edges. But this is not all: the crest on the back had
also lost its ragged appearance, thus conforming with the outline
of the new dorsal rim of the tail (Fig. to). It is not shed or
resorbed, but keeps the height of the courtship crest.

The object of my paper is to emphasize the similarity of these
processes of regeneration, reduction and compensation, in lower
and higher animal forms, and their relation to the reéstablishment
of the equilibrium of animal form.

Equilibrium of Animal Form 263

FIG.Y. Fie. /0.

264 Hans Przibram

LITERATURE

Emme L, V. E.—Regeneration and the question of symmetry in the big claws of
the lobster, Science, n. s., xxvi, 19, vil, 1907 (with literature).

Kammerer, P.—Regeneration sekundarer Sexualcharaktere bei den Amphibien.
Archiv f. Entwicklungsmechanik, xxv, 1907.

Mecusar, F.—Die Regeneration der Koleopteren. A. f. Entwm., xxv, 1907.

Morcan, T. H.—Notes on Regeneration. (Transpositional or compensator-
regeneration of the large chela in some Crustacea, p. 169). Bio-
logical Bulletin, vi, 1904.

Przizram, H.—Die “Scherenumkehr” bei dekapoden Crustaceen, zugleich: Experi-
mentelle Studien tiber Regeneration. Archiv. f. Entwicklungs-
mechanik, xxv, 1907 (with literature).

Witson, E. B.—Reversal of asymmetry in Alpheus heterochelis. Biological Bul-
letin, iv, 1903.

ZeELENY, CH.—Compensatory regulation. Journal of Experimental Zodlogy, ii,
1905 (with literature).

Zueizer, M.—Ueber den Einflus der Regeneration auf die Wachstumsgeschwindig-
keit von Asellus aquaticus, <A. f. Entwm., xxv, 1907.

THE EFFECT OF DEGREE OF INJURY, SUCCESSIVE
INJURY AND FUNCTIONAL ACTIVITY UPON RE-
GENERATION IN THE SCYPHOMEDUSAN, CAS-
SIOPEA XAMACHANA?

BY
CHARLES ZELENY

Wirn Four Ficurrs

INTRODUCTION

The present study is a part of a series of experiments whose
object is the investigation of some of the internal factors control-
ling regeneration in several representative forms. ‘The factors
taken up in Cassiopea are the degree of injury, successive removal
of a part and rhythmical pulsation of the disk. It is found that
removal of six of the eight oral arms constitutes the most favorable
degree of injury for the regeneration of each arm, and that from
this optimum there is a decrease in both directions. ‘The data for
successive injury show a greater rate of regeneration of the margin
of the disk after the second removal, than after the first. A com-
parison of the rate of regeneration of the margin in cases where
the disk was made to pulsate rhythmically with cases without
pulsation shows no advantage in favor of the pulsating ones, but
rather a retardation.

Contributions from the Zodlogical Laboratory of Indiana University. No. 92.

I am indebted to the Carnegie Institution of Washington, for the privilege of working at their labora-
tory at Tortugas. To Dr. A. G. Mayer, the director, I am under obligation for many kindnesses and
especially for suggestions in connection with the work.

The present paper is the fourth of a series dealing with the internal factors controlling the rate of
regeneration. The other three papers are: a A study of the rate of regeneration of the arms in the
brittle-star, Ophioglypha lacertosa, Biological Bulletin, vol. vi, no. 1, December, |1903; b The relation
of the degree of injury to the rate of regeneration, Journal of 'Experimental Zodlogy, vol. ii, no. 3,
August, 1905; c Some internal factors connected with the regeneration of the chele in the Gulf-weed

Crab, Portunus Sayi. In press. Carnegie Institution.

Tue JourNnar or ExreriMENTAL ZOOLOGY, VOL. V, NO. 2.

266 Charles Zeleny

METHOD

An abundance of material of all sizes was obtained in the moat
of the fort at Tortugas. The animals were found to live very well
in glass dishes in the laboratory with but a single change of water
each day. Every effort was made to keep all conditions, except
the ones used in comparison, as much alike as possible. None
of the animals were fed during the experiments and as a result
both normal and mutilated specimens decreased in size. Not-
withstanding this decrease the animals remained healthy and
regenerated readily even after severe injuries. The starving of
the animals was necessary because it is impossible to feed equal
amounts to animals with different degrees of mutilation.

DATA AND RESULTS
He The Influence of Degree of Injury on the Rate of Regeneration

The rate of regeneration of a single oral arm in each of the
following cases was determined:

a one arm removed at its base;

b two arms removed at their bases;

c four arms removed at their bases;

d six arms removed at their bases;

e all eight arms removed at their bases;

7 whole mouth apparatus removed.

Case 7 is not an integral part of the series because the arms do
not regenerate from the same level as in cases a to e.

Five groups each similar to the above were obtained, the mem-
bers of a group being approximately equal in size.

The data as given in Table I show that the rate of regeneration
of an oral arm is the lowest when that arm alone is removed.
From this minimum the rate increases up to the optimum at six
removed arms. It then decreases being less when eight arms are
removed. ‘The first five cases in the table are strictly comparable
because in each the arms were removed at the base, and therefore,
the regeneration is from the same surface in each. In the sixth
or last case f this is not true. Here the whole mouth apparatus,
including the arms, was removed and regeneration is not from

Regeneration in Cassiopea 267
8

the same surface as before. “The measurements were made after
death. The animals were stupefied in CO, sea-water and pre-
served in 10 per cent formalin.

TABLE I
——— eee : -_ ee ———
| | | WHoLE
One arm | Twoarms | Fourarms | SIX ARMS EIGHT ARMS MOUTH
APPARATUS*
l a l |
| bs Z a | 2}
| =I S| 8 q & | E |
| § A : hea e
} 5 | . .
: : : 5 : 4 rales S a ee
Behan! sea Bal na: E| al] e gee). | 8) 3
as RB =| a rs aS lees » Fe ay a 4 Soelieecs a
Sais STS Hime EOS Tete) Varo eeu AMO: | eteon| w| &
iS) Siw lo 3 4 = CI = 3 ? ae} gio g
= hee heal tl fea hoe ech | alate Te) Pelle Peeters oe pas
a) oD | os > a. +4 > rat 7 cect ot
lel | ONES et sca We (et cot OA: eet? eae eee ices len
|

Group A 10.5) -048| 8.4/1.5 | r79len.2 2.
-059/21.0,2.5 |.119/15.5/3.
-080/30.5|2.5 |.082/24.0/2.

-034.29.01 .75| .060 35-014.

© |.185|10.8|1.6 |.148
|.194 16.5/2.5 -I51|17.5|2-5 |.143 16.5|2.0 |.121
104 28.5 3.5 +123]25.0|2.5 |-100/25.0]1.5 |.060
/-134 26.02.85 .110|28.0/2.0 | .072|28 .0|2.15].077

-071/42.0/5.0 |.119/36.014. esas -122/34.5]3-0 | .087 32.5/5-0 154
|
| | |

.66 .058 \2.65|.212 3.241.144 13+171-157| 2.40|.117 2.45|.212

Group B 17.0|1.

GroupC 22. 5|1.
GroupD 29.01.

00 won
oxrnoo

Group EE |42.0)3.

Average

* Not strictly comparable with the others because the regenerating surface is at a deeper level.

The average lengths of the regenerated arms are 1.66 mm. for
the individuals with one removed arm, 2.65 for those with two
removed arms, 3.24 for those with four, 3.17 for those with six,
2.40 for those with eight and 2.45 for those with the whole mouth
apparatus removed. Since the individuals of a group are not
exactly alike, and since the amount of regeneration is dependent
on the size, the specific amount, 7. ¢., the regenerated length
divided by the disk diameter was obtained in each case. The
average specific amount of regeneration for each degree of injury
is given in Table Lin italic type. It is seen that from a minimum
for the case with the lowest injury it increases to an optimum when
six arms are removed, beyond which it again decreases. ‘Thus
with one removed arm the specific amount is .058, with two arms
removed it is .112, with four .144, with six .157, and with eight
ie

A series of individuals with different extents of removed mar-

268 Charles Zeleny

gins was studied with respect to the question of relation of degree
of injury to the rate of regeneration, but because of distortions
involving the whole umbrellar region, no adequate data were
obtained.

2 The Effect of Successive Injury on the Rate of Regeneration

The study of successive injury was confined to the margins.
As in the last mentioned case there was considerable trouble with
distortion of the disk. Eight individuals however were without
distortion and could be used for the present purposes. The
experiments on successive injury come under two heads. In one
series a number of individuals of a size were chosen. In half of
these the whole margin was removed and allowed to regenerate for
twenty-nine days, at the end of which time it had nearly completed
its regeneration. ‘This margin was then removed for the second
time at the same hour that it was removed for the first time in the
others. After twelve days the animals were killed and a measure-
ment of the new margins gave a direct comparison of first and
second regeneration as shown in Table II.

TABLE II

Width of regenerated margin in millimeters
First regeneration Second regeneration
a 8
2 1.6

Average... .:... 3 1.2

The second regeneration shows a decided advantage over the first.

In a second series the first and second regenerations were com-
pared within single individuals. A part of the margin of each
individual was removed, and after it had nearly completed its
regeneration It was removed again at the same time that a similar
segment from another part of the circumference was removed
for the first time. A direct elimination of indviduality was thus
obtained.

On account of individual differences in the method each case
is described separately. In each, however, the first operation

Regeneration in Cassiopea 269

came on June 17, and the second on July 16, twenty-nine days
later. Twelve days were allowed after the second operation.

In individual A about one-sixth of the margin was removed.
The regenerated margin was 2.0 mm. in width twenty-nine days
later. It was then removed a second time and a similar segment
was removed from a part of the circumference which had not been
injured. Twelve days later the animal was killed and showed
a second regeneration width equal to 1.8 mm. anda first regenera-
tion width of 1.6 mm.

Fic.1 Fic. 2

Fig. 1 Outline of disk of Cassiopea xamachana in which segment B is a second regeneration and
segment A a first regeneration of the margin.

Fig. 2 Outline of the disk of Cassiopea xamachana in which the unbroken line between B’ and
lower A is a second regeneration of the margin to be compared with a first regeneration shown between
B and lower A.

In individual B the character of the operation was the same as
in A but the removed portion of the margin was one-fourth of the
whole for each regeneration, and the fepenerced margin was 2.7
mm. wide as compared with the feared 4.2 mm. ie this case
the second regeneration width is 2.0 mm., and the first 1.1 mm.
The relative widths are shown in Fig. 1, where A represents the
first regeneration and B the second.

In individual C as shown in Fig. 2 in semidiagrammatic form
the original outer circumference is shown by the dotted line and

270 Charles Zeleny

outer unbroken line between B and A. At the first operation the
half of the margin shown in the figure by the part to the right of a
line between 4 and Ad’ was removed. This was allowed to regen-
erate until it had a width of 3.5 mm. as compared with 4.5 mm.
in the uninjured margin. The second operation was then made
and consisted of a cut along the heavy line between B and B’
including five-sixths of the whole margin. This second operation
included a strip of regenerated margin, and anequal strip of margin
which had previously been uninjured. After twelve days the animal

Fic. 3 Fic. 4

Fig. 3 Regeneration of inner and outer margins in a non-pulsating individual of Cassiopea xama-
chana. Compare with Fig. 4.
Fig. 4 Regeneration of inner and outer margins in a pulsating individual of Cassiopea xamachana.

Compare with Fig. 3.

was killed and the average width of the second regeneration
(B’ to lower 4) was 1.4 mm. as compared with a first regenera-
tion width (B to lower 4) of .8 mm.

In all three individuals there is a well marked difference between
the second and the first regeneration in favorof theformer. The
character of the operation to a very large extent eliminates chance
of individual error, and makes the result reliable. The five
individuals of the other series strengthen the conclusion that in
Cassiopea a second regeneration of the margin occurs more rap-
idly than the first.

Regeneration in Cassiopea PL 7/ A

The Effect of Rhythmical Pulsation of the Disk on the Rate of

Regeneration
f

I am indebted to Dr. A. G. Mayer for the method of obtaining
a rhythmically pulsating disk. The margin with Its sense organs
and the mouth apparatus is removed and the ventral surface is
cut as shown in Figs. 3 and 4. Such a disk remains quiet if
undisturbed, but a rhythmical pulsation can be produced by an
electrical stimulation at X. This pulsation in some cases con-
tinues for several days after a single stimulation. In an animal
operated on in this way there are two regenerating surfaces, an
outer one to replace the sensory margin and an inner one to replace
the mouth apparatus. In my experiments a comparison was
made between those with pulsating and those without pulsating
disks, to see whether pulsation has any effect on the rate of regen-
eration.

TABLE III
WIDTH OF REGENERATED WIDTH OF REGENERATED
INNER MARGIN OUTER MARGIN Duration of Duration of pul-
— = — regeneration sation in stimu-
Stimulated Unstimulated | Stimulated Unstimulated in days lated individuals
individual individual individual individual
1.0 2.5 aly 1.0 5 3 days
1.6 2.3 obs 1.0 5 4% days
1.3 6.0 6 A] 5 no pulsations
6.0 1.8 1.0 6 5 2 days
1.6 2.8 5 1.0 4 8-16 hours
2.8 2.8 1.0 1.0 4 8-16 hours
2.4 | 1.0 4 few minutes
1.2) | “5 4 no pulsations

Two factors must be considered as entering into the result of
the present experiment, first the stimulation of the animal as a
result of the electrical shock, and second the pulsation resulting
from this. It was very difficult to get pulsation that would con-
tinue for a considerable period of time. In all cases it stopped
before the completion of the experiment. The data are given in
Table III, which shows that the effect of the stimulation and
pulsation is inhibitory on the whole. In the table, as in the

Def Charles Zeleny

experiments, the individuals, except the last two are grouped in
pairs, one member of each pair being stimulated, and the other one
unstimulated.

In four of the six pairs of cases the unstimulated individuals
show a greater amount of regeneration than the stimulated ones.
In one the two are equal and in the sixth the stimulated is greater
than the unstimulated.

DISCUSSION

I The Relation of the Degree of Injury to the Rate of Regeneration

The results obtained in Cassiopea agree with the general rule
[ have found to hold true in the arms of Ophioglypha lacertosa and
the chela of Cambarus propinquus and which Ellis finds in the
legs of Mancasellus macrourus. The rate of regeneration of a
removed appendage is determined not only by the character
and position of the cut surface, but also by the character and
extent of other injuries received at the same time. The rate
increases with added injuries to other parts of the body up to an
optimum which represents the amount of injury most favorable for
regeneration. Beyond this point added injury causes a decline
in the rate of regeneration. In the case of the arms of Cassiopea
the optimum comes when six of the eight arms are removed.

2 The Relation of Successive In ury to the Rate of Regeneration
ee §

Careful investigations of the rate of regeneration after successive
injury are rare in the literature. [he general statement is, how-
ever, frequently made that the rate and character of regeneration
are unaffected by successive injury within very wide limits. I have
been able to find descriptions of three cases which do not agree
with this statement.

Vanlair finds that the sciatic nerve of the dog regenerates more
rapidly after the second than after the first removal.

Driesch finds in Tubularia that the development of the aboral
hydranth is more rapid after a second than after a first removal.
He also makes out an interesting case of effect of successive re-
moval on the character of regeneration in Antennularia. The free
basal end of the stem of this hydroid, after a first removal, develops

2 0 OY i , Sar 2 RD
Rege neration in Cassiopea 273

stolons alone. After a second removal it develops one or more
slender stems as well as the stolons, after a third removal two or
three strong stems and only one or a few slender stolons, and
finally after a fourth removal no stolons at all, the whole growth
consisting of one or two very stout stems.

To further test the question of the influence of successive
injury upon regeneration, I made a study of the problem in the
chelz of the Gulf-weed crab, Portunus Sayi, in the Scyphomedusan
Cassiopea xamachana and in several other forms. ‘The report
on the first of these is now in press, and the data show that while
the second regeneration is greater than the first, nevertheless, when
the age factor is eliminated the twoare exactly alike. In Cassiopea,
however, in which the age factor is also eliminated, the margin
shows in every case a greater rate of regeneration after the second
than after the first removal. The material on several other forms
is now being worked up.

3 The Relation of Functional Activity to the Rate of Regeneration

In view of recent discussions concerning the relation of form
regulation to functional activity a comparison of pulsating with
non-pulsating disks of Cassiopea is of special interest. Con-
trary to the general view that functional activity is an aid in
effecting form regulation, it was found that pulsating individuals,
with two exceptions, showed a slower rate of regeneration than
non-pulsating ones. The result indicates that there is need of
further investigation along this line before general conclusions
are made.

274 Charles Zeleny

LITERATURE LIST

Driescu, H., ’97—Studien tiber das Regulationsvermogen der Organismen. I.
Von den regulativen Wachstums—und _ Differenzirungsfahig-
keiten der Tubularia. p. 393, Archiv. f. Entw. Mech. d. Organis-
men, Bd. v.

Exris, M. M., ’07—The influence of the amount of injury upon the rate and
amount of regeneration in Mancasellus macrourus (Garman).
Biol. Bull., vol. xiii, no. 3.

Mayer, A. G., ’06—Rhythmical pulsationin Scyphomeduse. Carnegie Institution
of Washington, Publication no. 47.

Vantalr, '94—Archives de Physiologie normale et pathologique.

Zeveny, C., ’°03—A study of the rate of regeneration of the arms in the brittle-
star, Ophioglypha lacertosa. Biol. Bull., vol. vi, no. 1.

o5—The relation of the degree of injury to the rate of regeneration.

Jour. of Exp. Zodl., vol. ii, no. 3.

In Press. Some internal factors connected with the regeneration of the

chela in the Gulf-weed Crab, Portunus Sayi. Carnegie Insti-
tution of Washington.

STUDIES IN ADAPTATION
I THE SENSE OF SIGHT IN SPIDERS

BY
ALEXANDER PETRUNKEVITCH, Ph.D

With Six PrLates

Whether the organization of an animal is the result of contin-
uous adaptation to the conditions of life, or whether the animal
adapts itself as best it may to the use of organs in the possession
of which it has been put by nature, an impartial observer sees
everywhere the remarkable balance between structure and function.
The few apparent incongruities which we meet with in all groups
of the animal and vegetal kingdoms, only serve to confirm the
rule, since they are due lareely © to our lack of knowledge of the
conditions under which such organs exist to their best eee
or of the role which they played in the phylogenetic development
of the species. But however this may be, we should expect to
find, and there surely must be a marked difference between the
origin of organs by means of which the animal communicates
actively with the surrounding world and that of such organs or
structures as lie entirely outside of the personal activity or con-
trol of the animal. It 1s very probable that many, perhaps the
majority of arthropods are entirely unaware of the presence on
their bodies of so-called decorative structures, designs or colors
and whether a male possessing a slight variation in such a
structure, design or color, will or will not be preferred by a
female, is to my mind entirely a matter of chance. In this con-
nection I can only confirm the observation of earlier naturalists
that females often choose defective males in the presence of
other in every respect perfect ones. I have caught in Europe a
pair of large beetles (Prionus coriarius), in coitu in the presence
of two other perfect males, when the male in question had

Tue JourNar oF ExpertMENTAL ZOOLOGY, VOL. V, NO. 2.

276 Alexander Petrunkevitch

one eliter and one leg malformed and showed other minor
defects. Upon investigation the male proved to have normal
spermatozoa and the female laid an extraordinarily large number
of eggs. Similar cases are common among all insects as well as
other animals. I have made the same observation in the beetle,
Lucanus cervus, the males of which have the beautiful horns on
their heads. Of the beautiful moth, Samia cecropia, over a hun-
dred of which I allowed to develop in a cage for other experi-
ments, many couples with entirely defective and malformed wings’
were found in coitu. Among orb-weaving spiders are several
species in which during the mating period the female allows
several males to remain in her net, of course at a good distance
from her, and it is a common occurrence for defective males to be
accepted by the female while perfect ones will be chased away for
their dear life or will even fall victims to the voracity of the much
stronger female. Protective structure or coloration belongs to
the same group of phenomena and here a great number of cases
might be recorded where an animal remains entirely unaware of
the protection afforded by its form and color if in the proper sur-
soundings and of the danger of disregarding this. I have seen
butterflies that would be protected by the leaf-like coloration of
their wings if they should sit motionless, instantly caught on the
wing by large dragonflies, and in the West Indies I could observe
regularly a hemipteron of the group Emesidw, which would
resemble a dead twig if in an appropriate position, swinging to
and fro on the four hind legs in the center of a large leaf. All
this I bring here by no means with the object of denying the
existence of adaptation or of the principle of selection itself, which
only one who has never observed nature outside of the laboratory
can do. But I want to show that in all those instances where the
animal itself cannot make use of a new variation or mutation, in
the possession of which it has been placed by nature, its advanta-
geous character will be a mere matter of chance, and selection, if it
take place, must be greatly retarded in its progress.

Quite different must it be with those structures which the animal
is able to put to active use, however unconscious it may be of the
advantage it enjoys over other individuals of the same species.

The Sense of Sight in Spiders 277,

A caterpillar that is able to construct a better protected cocoon
than its fellows, an animal that can run faster from danger, a
bird of prey that distinguishes its victim at a greater distance, all
these will evince their superiority over other individuals of the
same species upon the first occasion. Here, then, opportunity is
given for a more rapid accumulation of characters and an acceler-
ated selection. Several years ago I studied, together with Dr.
von Guaita, although with a different object in view, the stridu-
lating organs of One These organs are undoubtedly not
of a protective character as the insects in question are safer when
silent. This is sufficiently shown by the fact that they stop their
cry instantly upon the approach of danger. The origin of the
stridulating organs has therefore to be sought in some advantage
which they may afford the sexes during the mating period. That
they are a rather late acquisition is evidenced by their late appear-
ance, only with the last moulting, while the rapidity of selection
which in this case must still have been retarded by the danger
arising from their possession, is apparent from the complete
absence of any traces of similar organs previous to the last moult-
ing. The great variability in the number of teeth on the chord or
bow of the stridulating apparatus, which permits of difference of
pitch in the tones produced by friction, the fact, with other words,
that the stridulating organs are in respect to their details, not
absolutely fixed structures, seems to be due to this antagonism
between the advantage afforded by the organs in the relavon of
the sexes toward aan other and their dieade antage In respect to
diminished safety from the aggression of enemies. But how is it
with organs in which the advantage arising from their perfecting
will be immediately exploited to its fullest extent by the animal
possessing them? With this question before me I commenced
my research on the sense of sight in spiders. The results of this
research which now extends over more than a year, | bring in this

paper.

Two factors in the life of spiders have left a deep impres-
sion on their organization, the first, that they subsist exclusively
by means of prey and the second, that the external sexual organs,

278 Alexander Petrunkevitch

the organs of copulation in the male, are entirely separate from
the internal sexual organs. If we may judge of the amount of
food required by the quantity of insects captured in the orb-net
of a single spider, a quantity that is sometimes appalling, it was
necessary for the spiders, in order to satisfy this want, to develop
as they indeed have done, either the instinct and the engineering
capacity for constructing nets or else the instinct for hunting their
prey freely on the ground or on plants. ‘Two different directions
are thus given to the development of the whole spider group and
we should naturally expect to find differences in the structure of
the corresponding organs of sense and of the spinning apparatus.
But while in higher animals, as for example, birds of prey, all
organs of sense are developed to a remarkable degree of perfection,
we cannot say the same for the hunting spiders. It is a matter
of common observation that vultures discern their prey from a
distance amounting at times to several thousand feet and they
are undoubtedly able to scent a carcass hidden among bushes
at a considerable if not so great a distance as this. Their ears
are sensitive to high and low sounds of a very small amplitude
and only the sense of touch which could scarcely be of use to them,
is little developed. It is otherwise with the hunting spiders.
They possess doubtless, a very fine sense of touch, the whole body
being covered with hairs and bristles sensitive to the slightest
stimulus. In regard, however, to the organs of hearing and
smell we are as yet without definite results of any kind. At any
rate these two senses are very little developed. Even the ques-
tion as to the existence of such organs is to my mind far from
being settled. Whether the organs discovered by Dahl and the
lyriform organs have anything to do with these senses must be
determined by new investigation. I myself have made some
experiments on the sense of hearing, thus far without any definite
results. I alsorepeated the experiments of Pritchett on the sense
of smell in large lycosids but even to such irritants as formalde-
hyde, osmic acid and acetic acid, they did not respond soreadily or
so quickly as to the slightest touch with the end of a silk thread.
As the sense of touch 1s solely protective, there remains only the
sense of sight to guide the spiders on their hunting trips. The

The Sense of Sight in Spiders 279

splendid experiments of Mr. and Mrs. Peckham on the jumping
spiders (Attide), during the mating period, have clearly demon-
strated that the sexes recognize each other by the use of their eyes
alone, the male remaining unaware of the presence of a female,
nor will he perform the peculiar, characteristic love dance, if his
eyes are covered with paint. But is the sense of sight in spiders
as sharp as we should be led to expect by comparison with that
of birds of prey f This question has not yet been answered with
sufficient certainty owing to the difficulty of experimenting,
Plateau came to an enacts negative conclusion, asserting that
spiders possess an indistinct and very poor vision, being unable
to discern objects beyond a distance of from 8 to 10 cm. Forel
was of the same opinion, while the more ingenious experiments of
the Peckhams leave no doubt that the males of the Attids recog-
nize their females at a distance of about 30 cm. and moreover
that they distinguish colors. But whether spiders can see beyond
this distance and how sharp their vision is, the experiments of
the Peckhams also, leave unanswered. In fact it would be impos-
sible to answer this question merely by observing the behavior
of spiders during an experiment or in nature. A close anatomico-
physiological study is required and only by combining the experi-
ment on the living specimen with its after examination may one
reach a satisfactory answer. [This answer I hope to have given
in the present research.

Keeping in mind all that has been said in the preceding pages,
we may conclude that the study of the eyes of spiders and of their
sense of sight, as examples of adaptation of the first kind, pos-
sesses several advantages as well as certain disadvantages and
these we have next to consider. “The advantages are:

1 That the sense of sight is beyond any Spake the only sense
that guides hunting sniders on their hunting excursions and in
finding the females during the mating period.

2 That the eyes of spiders are organs which are for each species
definite in number and position on the cephalothorax.

That the sense of sight is a sense common to the great
majority of lower and higher animals and that some analogizing
is therefore not only admissible but may be of great value.

280 Alexander Petrunkevitch

The disadvantages are:

1 That beyond some experiments of Plateau and the Peck-
hams, nothing definite is known in regard to the acuity of vision
in spiders and no method has been brought forward for its study.

2 That the eyes are complicated organs, consisting of a refrac-
tion and a perception apparatus, each of which and the separate
parts of which may possibly be capable of adaptation and which
have therefore to be considered separately.

Let us now begin with a closer study of the ocular group on the
cephalothorax of various adult spiders.

THE POSITION OF THE EYES ON THE CEPHALOTHORAX

The number of eyes in the great majority of spiders is eight
and the group which they form on the cephalothorax is so
characteristic for the different families that it was for a long time
used as a systematic character of great value. The group con-
sists usually of two or three rows, more rarely of two or three
smaller groups containing three or two eyes each. Of importance
for systematics are the length of each row, the distances between
the eyes taken in relation to their diameters, the form of the row,
whether straight or bent in the middle forward or backward, as
well as the shape and to some extent the color of the eyes. Even
a superficial observer will notice in addition, that both in the
jumping and the ground spiders (Attida and Lycoside), two of
the eyes on the forehead are larger than the others, especially in
the former. But the reason for such a configuration in the eye-
group was never sought for and the apparent similarity in the
eye-groups of spiders belonging undoubtedly to different families,
led to the conclusion that the value of the eye-group as a sys-
tematic character had been overestimated.

The position of the eyes on the cephalothorax becomes more
comprehensible when we begin to study the directions of their
respective axes and the angles that these axes form with the three
chief planes of the body. Unluckily we at once meet with great
difficulties even though we choose species having perfectly round
eyes. ‘The first of these is to ascertain with exactitude the posi-

The Sense of Sight in Spiders 281

tion of the two planes intersecting the plane of symmetry of the
body. After experimenting for a long time I decided upon the
following method. The spider is killed in alcohol and then kept
in it for several days. When the muscles have become entirely
rigid, so as to allow of no change in the shape of the cephalo-
thorax while drying, the abdomen is severed from the cephalo-
thorax through the petiolus with a sharp pair of scissors. Next
the pars labialis, together with the laminz, the palpi and the chela,
is carefully removed with forceps. At the same time care must
be taken not to tear the chitinous bridge connecting the opposite
sides of the cephalothorax immediately behind the chelz since
this would lead to a flattening of the cephalic part and consequent
distortion of the true angles. ‘The legs are now carefully removed
leaving the cox alone in their normal position attached to the
sternum. Next a thin line is drawn with chinese ink on a very
thin layer of Canada balsam that has been spread with the finger
over a small cover-glass. It is best to use rather a thick cover-
glass and to draw the line quite across it parallel to two edges.
A drop of fish glue is now put in the center of the glass and on this
the cephalothorax is placed with the sternum toward the glue and
gently pressed until all the cox and the sternum are in contact
with the glass. The glass now represents one of the two planes
intersecting the plane of symmetry at right angles. I shall call
it the horizontal or foundation plane. It is only approximately
parallel to the surface of the earth or to that of any object upon
which the spider may be, since the spider is able to raise itself up
on either front or hind legs, in this way changing the angle between
the foundation plane and the horizon. At the same time it is
essential that the plane of symmetry should coincide with the
black line on the cover-glass. This is accomplished by using
a needle under the microscope at a magnifying power of about
twenty diameters. It is now easy to determine the plane of sym-
metry by taking the point midway between the front middle eyes,
the central, longitudinal groove of the cephalothorax when it is a
species possessing this groove, and the point at an even distance
between the two chitinous plates of that part of the petiolus which
remains with the cephalothorax after the abdomen has been

282 Alexander Petrunkevitch

severed. The third plane which I call the vertical or transverse
plane and which intersects the two other planes at right angles, is
geometrically determined by these. After the cephalothorax has
been prepared in this manner and thoroughly dried in a warm
place, the cover-glass upon which it is fixed is laid upon the center
of a square glass plate, which is at the same time the center of a
circle drawn upon the surface of the plate with radii forming angles
each of which measures ten degrees. If we now use an eye-piece
having two lines intersecting in the center at go°, we can readily
measure the angles that the eye-axes form with the plane of sym-
metry. To know how this is done we must remember that the
eyes of spiders have each a lens, the outer surface of which forms
part of the surface of a sphere. When we look at a lens in the
direction of the eye-axis, it appears to us as a circle while if we
look as it under an angle of less than go°, its outer surface appears
to us as two curves intersecting each other at two points. If we
place the eye in the center of the microscopic field and move the
eye-piece until one of its lines falls upon these two points of inter-
section of the curves of the lens and the other passes through a
point midway between them, then the latter line represents the
projection of the eye-axis on the horizontal or foundation plane
and the angle that it forms with the plane of symmetry can be
read directly from the scale. More exact results would of course
be obtained by using a goniometer ocular such as is made by
Zeiss, but even the arrangement I have described here, the ocular
with intersecting lines, gives an error of not more than a few
(3 to 4) degrees. When the angles have in this way been measured
a drawing is made with the aid of an Abbe drawing apparatus
of the entire eye-group and the eye-axes are then drawn in, in
accordance with the data given by the measurements. We obtain
in this way a correct figure of the projection of the eye-group on
the horizontal or foundation plane. In order to gain a clear pic-
ture of the position of the eyes it is necessary to make two other
projections, one on the plane of symmetry and the other on the
vertical plane. ‘Lo accomplish this I put a small drop of beeswax
in the center of the circle on the glass plate into which the edge
of the cover-glass holding the cephalothorax is pressed. This

The Sense of Sight in Spiders 283

cover-glass is held in a vertical position with the aid of two straight
angles which are removed as soon as the wax is hard enough
to keep the glass in position. Another method, much simpler
and perhaps just as good, consists in adjusting the cover-glass
until its upright edge forms a straight line with its own reflection in
the slide. The angles are now measured in the same way as
before. The drawing is of course made under the same magnif-
cation and the upper edge of the glass holding the cephalothorax
is likewise drawn. It represents a cross-section through the
horizontal or foundation plane.

To make a drawing of the projection on the vertical plane one
proceeds in the same manner with that difference that the cover-
glass carrying the cephalothorax is placed on its other edge.

Two facts become immediately apparent upon comparing the
drawings made in this way, of eyes of spiders belonging to
different families. First, that there is not a single pair of eyes
which are focused upon a single point like, for instance, the eyes
of man. On.the contrary, the axes of all eight eyes are so directed
as to form divergent angles with each other. Second, that not only
do the positions of the eyes on the cephalothorax of spiders belong-
ing to different families differ from each other, but the axes of the
same eyes in different spiders do not lie in the same direction but
form with the three planes of the body, the planes of projection,
angles differing considerably from each other but fixed for each
species. Yo make this clear let us look at the drawings (Figs.
1-9) representing the eyes of three hunting spiders, Lycosa
nidicola—a common large ground spider of morhenn America,
Phidippus tripunctatus—a large ] jumping spider belonging to the
same region and Heteropoda venatoria—a samiGnsien fropiel
and Sricecel spider of very large size, belonging to the family
Heteropodidz and resembling i in habitus the crab spiders (Thomis-
idz) among which it was Saat placed by earlier systematists.

Translating the results of these measurements into common
language, we may thus describe the positions of the eyes in the
three spiders. The anterior middle eyes in the jumping spider,
Phidippus tripunctatus, are directed forward and a little outward
and downward. In Lycosa nidicola more outward and consider-

284 Alexander Petrunkevitch

ably upward. In this respect Heteropoda venatoria resembles
Lycosa more than it does Phidippus, since its anterior middle
eyes are also directed frontward but still more outward and con-
siderably upward although not so much so as in Lycosa.

TABLE I
Projection on the horizontal or foundation plane. S e Figs. 1,2 and 3
The angles which the axes of the eyes form with the plane of symmetry. The right side with a plus, the

left with a minus sign

AME | ASE PME PSE

Phidippus tripunctatus.......-......... | Se 8 ZI 67 95

MY cosaynidicol acim jer (e cpese csc erelater-lelesniste | a= 12 24 24 94

Heteropoda venatoria...............+0- + 32 27 ° | 109
TABLE II

Projection on the transverse or vertical plane. See Figs. 6,8 and Q

The angles which the axes form with the plane of symmetry. Signs as before

AME ASE | PME | PSE
Phidippus tripunctatus..............+-. ae P 57 | 63
EY.COSaLDIAICO] ay terete ators ie) =1=:¢Fo sie l=|= cisisiets s ae ; 155 ? 56
Heteropoda venatoria.............+.++. | Be ? 8 | 71

TABLE Ul
Projection on the plane of symmetry. See Figs. 4, 5 and 7
The angles which the axes of the eyes form with the horizontal or foundation plane. Zero in front of the

head, 180° at the back. Positive quantities for eyes looking upward, negative for those looking down-

The anterior side eyes in Phidippus are so directed that their
axes are parallel to the horizontal or foundation plane and turned
a little sidewise. In Lycosa they are directed a little more out-
ward and at the same time downward. In Heteropoda they are
directed still more outward but at the same time upward.

The Sense of Sight in Spiders 285

The posterior middle eyes in Phidippus are directed consider-
ably toward the side and upward. In Lycosa they are directed
much more frontward and also upward, while in Heteropoda
they look straight upward. ‘Their axes are in this case almost
perpendicular to the horizontal plane.

The posterior side eyes in Phidippus are directed sidewise,
considerably upward and a little backward. In Lycosa they are
directed in the same way sidewise and backward but considerably
more upward, while in Heteropoda they are directed considerably
less upward and much more backward.

I studied the positions of the eyes as I have described them here,
originally in perfectly ripe females. Six individuals of each
species were measured and showed only slight differences in the
angles. Since the method employed is not an absolutely exact
one, it is difficult to say whether these differences depend upon
variation in the position of the eyes or upon defects in the method
itself. However, the following facts speak rather for defective
measurements than for natural variation. I expected to find
that during the post-embryonic development the position of the
eyes on the cephalothorax would change with each moulting,
approaching more and more nearly to that of the adult female,
which should be considered an adaptation to the particular_life
of the spider. An observation that made this seem still more
probable has been made by various scientists at different times,
that the eye-group in young spiderlings occupies a relatively
larger part of the qoute oes: than it does in the adult spiders.
Nevertheless there seemed to me to be occasion for a more thorough
study of the eye-group and the eye-axes in spiders of different
ages. I had in my possession perfectly ripe females of the
three species I have mentioned, several specimens of Heteropoda
just before the final moulting, others that had to moult twice and
three specimens of very small spiders that had still to moult at least
three times before attaining maturity and also a cocoon filled with
very young spiderlings. I kept sev eral females of Lycosa nidicola
for a time in large glass jars and preserved both mother and the
spiderlings which were in part taken from the cocoon, in part
killed while on the back of the mother just as they were about to

286 Alexander Petrunkevitch

leave her. I had, besides, several unripe specimens of unknown
age. I had also Phidippus in the same stages as Lycosa. It 1s
not difficult to obtain these since the female of this species make
a tent of web in which she lays the eggs, afterward guarding the
young ones for a considerable time. | caught in addition several
females of Pardosa nigropalpis with young ones on the back.

The method employed is as follows: Instead of measuring each
eye and the distances between the eight eyes in each spider, I make
with the aid of the Abbe apparatus a drawing of the entire eye-group
of an adult female. Leaving the drawing in the same position
on the drawing table, | remove the adult female and substitute a
younger one. I then try different objectives and oculars until
the image on the paper of the eye-group of the younger spider is
of the same size as the drawing of the adult. A sheet of clean
paper is now put in place of the one with the drawing and the new
drawing is made. In this way drawings are obtained of all
stages. The angles of the eye-axes are next measured and the
axes drawn in on the corresponding figures. In all cases, begin-
ning with the young spider at the time when it is ready to leave
the mother in order to commence its own, independent existence
and ending with the mother herself, the configuration of the eye-
group and the angles of the axes proved to be the same and the
drawings made of them on paper, when superposed and examined
against the light, coincide absolutely. But this does not apply
to the youngest spiderlings, those taken directly out of the cocoon.
Although in such spiderlings the eye-group is in general very
nearly the same as in the adults, careful measurements show dif-
ferences which, while not appreciable to the unaided human eye,
are nevertheless of great importance. I give these measurements
here (Table [V) but shall discuss them farther on when exam-
ination of the fields of vision will reveal more clearly their
significance. It is unfortunately still more difficult to measure
the angles in such spiders than it is in older ones so I give only
figures of which | am certain.

If we compare these tables with those for the adult females
we shall at once notice the following differences. In the youngest
spiderlings of Phiddippus and Lycosa the anterior middle eyes

The Sense of Sight in Spiders 287

are directed a little more outward and in Phidippus a little more
downward also, than the same eyes in the adult. In Heteropoda,
on the contrary, the anterior middle eyes of the spiderling are
directed much more frontward than in the adult.

The anterior side eyes in Phidippus and Lycosa are directed
much more toward the side than in the adult, in Heteropoda much
more toward the front. The angles of projection on the two

TABLE IV
SHOWING THE ANGLES THAT THE AXES OF THE EYES FORM WITH THE THREE PLANES OF THE BODY IN
VERY YOUNG SPIDERLINGS

a Projection on the horizontal or foundation plane. Compare Figs. 4, 5 and 7

AME ASE PME PSE
Phidippus tripunctatus................. Be 10 30 ; 88
Tey COSAapNIGICOl aiatetsrasstetesayelo\sjevs/=\ele-=iche"= lai* | + p 30 25 85
Heteropoda venatoria.................. Be II 22 18 60

b Projection on the transverse or vertical plane. Compare Fig. 9

AME | ASE PME PSE

Phidippus tripunctatus................. ee ? ? ? ?
Wyycosamidicola'ejereroforcinvessi-\-)-ite)atsjej-isiere% se ; ? ; 68
Herteropoda-venatoniaz: «20.2.0. ose So ? ? ? ?

c Projection on the plane of symmetry. Compare Figs. 4, 5 and 7

= ———
AME ASE PME PSE

i}
Pinot pine tee ete eek tewe is = ° | ; ?
ycosamidicolacnaesscteaitictsts Seveciecte= P ? | + 30
(Hetero podaivenatortasjetatcicleisie + cstie'siciieieie ce ; ? =e 7 ?

other planes could not be ascertained for these eyes. It is impos-
sible to study the posterior middle eyes in the young spiderlings
of Phidippus at all, on account of their extreme minuteness. In
Lycosa the projections of the axes of these eyes on the horizontal
plane is approximately the same as in the adult but their pro-
jection on the plane of symmetry shows that they are directed
upward at an angle about three times as great as in the adult.

288 Alexander Petrunkevitch

In Heteropoda spiderlings they are directed somewhat sidewise
and nearly upward but more toward the front, while in the adult
they are directed straight upward and a little backward.

The posterior side eyes in the spiderlings of all three species,
especially in Heteropoda, are directed a little frontward instead
of backward and in Lycosa less upward, also, than in the adult.

The relative sizes of the eyes and of the distances between them
are also different for spiderling and adult. Tables V, VI, VII and
VIII may serve to illustrate this.

TABLE V
Lycosa nidicola. Diameter of eyes in millimeters

y f

| AME | ASE PME PSE
Moo thertage ict crctaaiete tate atte ere astars aptors | 0.361 | 0.279 | 0.689 0.541
Spiderling, ready to leave mother............ 0.074 | 0.057 | 0.148 0.115
Spiderling, taken out of acocoon........... ‘| 0.049 0.038 0.115 0.115

Or in proportion to the anterior middle eyes which we take as unit of comparison

| AME ASE | PME PSE

Mother Sakai. ceiccin tceats cin ieiere rote wie eres I | 0.77 1.90 | 1.50

Spiderling, ready to leave mother........... I 0.77 2.00 1.55

Spiderling, taken out of acocoon............ I 0.77 | 2.30 2.30
TABLE VI

Heteropoda venatoria. Diameter of eyes in millimeters

| Length of | | |

| cephalo- | AME | ASE PME ESE

| thorax
Adiultsterma lessees cyele ne lesei-eeynatera' te | 10.6 0.45 0.75 0.55 | 0.70
Immature female before last moulting....| 6.8 | 0.28 0.55 0.40 | 0.50
Two moultings before maturity.......... |

5-4 0.22 0:43 - 1OT gl aee

Or in proportion to the anterior middle eyes

| AME ASE | PME | PSE

|
AGN Manel Ds gone anese aeonenuaanpoenaadd I 1.66 1.22 Te55
Immature female before last moulting....... I | 1.93 | 1.43 1.78

Two moultings before maturity............. I | 1.95 1.54 | 2.04

The Sense of Sight in Spiders

289

SHOWING THE DISTANCES BETWEEN THE EYES AND BETWEEN THE EYES AND ThE EDGES OF THE

CEPHALOTHORAX

Lycosa nidicola. Measurements in millimeters

mes & Gis 4 | g28
s a 22 5 | eee
| cra o Bb o ae son
oO 8 ac) o 2107
a ‘34 we! % af
J
ok a5 $=2n 2 $yaes
ain 3S | a2 St ist Oo.
tS Se a bo $)2° oid
=e) 28 23a as Boos
oO eo
H aa 8 I a
Adultfemale rc ryatlerisie oleate 8.1 6.0 aie) Tear 2.2
Spiderling taken out of a
COCOOD. Hane jate-nioe che tietseyavel- 0.984 0.771 0.459 0.197 0.180
Or in proportion to the length of the cephalothorax as a unit
|
Adultstemalesmrrremtertctasrs I 0.74 0.27 0.13 0.27
Spiderling taken out of a
(elke coagapubeondvecnuK I ‘ 0.78 0.46 0.20 0.18
Heterapoda venatoria. Measurements in millimeters
INE Gn Alsace seouane on | 8.0 8.0 3-2 1.4 1.6
Spiderling taken out of a |
|
(Selsey GnoohogdacenroododG 1.017 ©.go2 0.525 0.180 0.131
Or in proportion to the length of the cephalothorax
; a“ 7 2
Adil teitern aleryeterecciareterstsie ave I Xie | 0.4 0.17 0.20
Spiderling taken out of a | |
COCGOMs seratata pistes terete hrcte I. 0.88 | 0.51 0.17 0.12

290 Alexander Petrunkevitch

By a comparison of all these tables we may now gain consider-
able light on the question as to what happens to cephalothorax
and eye-group during the post-embryonic development. When
the spiderling sheds its first skin in the cocoon, the eye-group
occupies almost the whole breadth of the cephalothorax which
is comparatively very low. The first change, of which I shall
speak farther on, is in the directions of the eye-axes. When the
spiderling leaves the cocoon, that is, before the next moulting takes
place, the eye-axes have become fixed in the positions which they
will occupy during the whole life of the growing and mature spider.
Whatever change takes place from the time the spiderling leaves
the cocoon, is only in the relative size of the space on the cephalo-
thorax occupied by the eye-group and to a certain extent, in the
relative sizes of the eyes themselves. The cephalothorax grows
more rapidly than the eye-group,so that the latter occupies with
each moulting a relatively smaller part of the cephalothorax.
We shall presently see that this also is of advantage to the spider.

THE MAXIMUM *ANGLE AND THE FIELDS OF VISION

While handling under the microscope a dried out cephalo-
thorax from which all organs and muscles had been removed to
permit of the study of the endoskeleton, I chanced to notice that
the faint images, visible in the eyes, of the trees which grow before
my laboratory window, were not of the same size. I very soon
found that boiling or even keeping in a cold solution of potassium
hydrate so changes the optical property of the eye-lens that it
becomes entirely intransparent so that it is necessary to use some
other method. I have finally adopted the following one. The
spider is killed in strong alcohol from which it is at once removed.
The abdomen, all appendices and the sternum are then removed
and the organs and muscles filling the cephalothorax are care-
fully taken out with a forceps. Next the inside of the cephalo-
thorax must be cleaned under water with a soft brush. I use a
small camel’s hair brush of the kind used for water colors but cut
the hairs quite short. With this brush it is possible to remove
all the remaining muscles as well as the vitreous bodies of the

The Sense of Sight in Spiders 291

eyes. Care must be taken, however, not to remove the black
pigment ring surrounding each lens on the inside surface of the
cephalothorax and forming a sort of iris, as this would make a
difference in the measurements. Even in spiders that have
been kept for a long time in alcohol the lenses are often still so
transparent that one may see the images formed by them, but such
lenses are yellow and the images rather poor. If on the contrary
the eyes are prepared in the manner just described, it is scarcely
possible to give an idea of the beauty of the little images. The
lenses are then entirely colorless and transparent and the images
render correctly color and line.

For the study of the fields of vision, each eye together with a
little of the surrounding chitin must now be cut out of the cephalo-
thorax. ‘The lens is then placed with its inner surface on a small
drop of liquid on a slide and in a hanging position examined under
the microscope through the slide. By this means three advan-
tages are gained. First, the object examined sends its rays
through the lens in the normal direction so that the eye of the
examiner is substituted for the retina of the spider; second, the
observer looks in the direction of the eye-axis or at least very
nearly in that direction; and third, the outside surface of the lens
remains dry, limited by the air alone, as is the case with the living
spider. More difficulty is presented by the fact that the refraction-
coefhcient of the vitreous body is not known. However it is
sometimes possible to prepare an eye fresh with the vitreous body
in its natural position and the retina cut off with the aid of a
razor. We are then able to measure the image of a scale at a
given distance. But since the vitreous body coagulates too
rapidly to be used in the study of the maximal and minimal
angles of vision, we have to use in its stead a drop of water and
also of some liquid possessing a higher refraction-coefhcient
than the vitreous body. With the latter we obtain a somewhat
larger image than in reality and may overestimate the acuity of
vision. Such a liquid I found in a mixture of equal parts of pure
glycerine and egg albumen. ‘The results obtained by the use of
water we may then employ for control, to guard us against the
opposite extreme of an underestimation of the acuity of vision.

292 Alexander Petrunkevitch

The proportion between the two media is according to my measure-
ments as 4 : 5, 7. ¢., that an image will occupy four divisions of a
scale if water is used as the medium of suspension as against five
divisions when glycerin-albumen is used.

The microscope also must be arranged in a special manner.
The diaphragms with the mirror and the Abbe lens must be
removed. ‘The instrument is then placed on a high box open to
the window, with a long slit occupying the whole space between
the legs of the stand. Next a scale in the form of a cross with
right angles is drawn on bristol board. Each arm of the cross
is two centimeters wide and consists of alternating black squares
like those on a checker-board. For the sake of convenience as
well as to avoid error, the numbers are written in each white
square in roman numerals in one direction and arabic in the
other. This cardboard is now placed under the microscope so
that the distance between it and the spider’s eye is exactly 10 cm.
Excessive light around the eye is excluded by means of a small
diaphragm or a black paper with a small round hole arranged so
that the spider’s eye hangs directly in the middle of the hole. Of
course any objective with small magnifying power may be used.
As for myself I either use the a* or the A achromatic system of
Zeiss and the compensation ocular 6 with the ocular micrometer.
This micrometer is adapted to apochromats but may just as well
be used with achromats if one ascertains the size of each division.
In my instrument each division of the micrometer with the
A objective corresponds to 0.0164 mm., while the correction of
the a* lens makes possible a magnification where each division
will correspond to 0.1 mm. .The light reflected from the white
bristol board on which the scale is made, is sufhcient to give a
perfect image of the scale in the microscope.

The scale is so placed that the center of the cross falls exactly
on the axis of the spider’s eye. | found that the eyes of the hunt-
ing spiders are quite round and that the maximum angle of vision
is therefore the same in each direction, 7. ¢., the limit of the field
of vision in such eyes 1s a circle representing the circumference
of the base of a cone. In order to find the maximum angle in
each case, there remains only to read on the image of the scale

The Sense of Sight in Spiders 293

in the eye, how many centimeters are visible. Since the dis-
tance between the spider’s eye and the scale equals 10 cm. a
simple calculation will give the value of the angle in question, or
it is still simpler and entirely sufficient for our purpose, to draw on
paper an isosceles triangle, the base of which must be as many
centimeters long as are visible on the image of the scale in the
eye and its height ro cm. ‘The angle can now be directly meas-
ured. When the angles have been measured for all the
eyes, they are represented on the drawings in each _ projection,
showing the field covered by each eye. Optically the angle
depends upon the curvature of the lens and the refractive coef-
ficient of the substance of which it consists. But what is of
interest for us here is the general fact that the larger the spider’s
eye, the smaller, as a rule, is its field of vision.

If we compare the drawings of corresponding projections in
different spiders after the maximum angles of vision have been
introduced, we cannot fail to recognize the remarkable relation
between the particular life of the spider and the position of its
eyes. In order to make this clear I must state here that which I
shall prove farther on, that the larger the spider’s eye, the sharper
Its vision or power of distinction. Let us begin with an examina-
tion of the projection on the horizontal or foundation plane (Fig.
1). We see that the largest eyes in Phidippus are the anterior
middle ones covering a field of 40° each or both together about
55°, owing to the fact that their axes are a little divergent. Each
of the anterior side eyes also covers a field of 40° or both together

3°, 1. e., more than the entire field covered by the AME! But
the ASE are considerably smaller than the AME. The minute
eyes of the second row, the posterior middle eyes, cover a field
of 62° and the PSE one of 48° or the whole eye-group covers
about 240° of the horizon. The projections on the other two
planes (Figs. 4 and 6) show in addition that the PME and the
PSE guard chiefly the sides of the spiders, leaving about 52° in
the vertical plane and more than 80° at the back on the dorsum,
entirely unguarded. ‘This is the only direction from which the

‘These are abbreviations commonly used by arachnologists. AME stands for anterior middle eyes;
ASE for anterior side eyes; PME for posterior middle eyes, and PSE for posterior side eyes.

2.94 Alexander Petrunkevitch

jumping spider can be taken unawares by an attacking enemy.
We know besides from the behavior of the spider that when-
ever an insect or anything else approaches it from the side, it
immediately turns toward the intruder as though with the
desire to see it better by using its front eyes.

Lycosa nidicola is a spider that lives on the ground under
stones, making excursions in the grass. Its manner of walking
like that of all ground spiders, is distinctly straight forward and
we find that the largest eyes, the posterior middle eyes, are so
situated as to guard the front of the animal. In the projection
on the horizontal (Fig. 2) plane they together cover a field of 48°,
1. e., considerably less than the four eyes of the front row, which
cover all together a field of 77°. Between the eyes of the second
and those of the third row there is an unprotected area of
about 7°, or remembering that the drawing is considerably
enlarged, we may say that an object I cm. sq. will be invisible
within the space of these 7° as soon as it is farther than 8 cm.
from the spider. The presence within this area of a spider of the
same species could be already noticed at a distance of about 25
cm., quite sufficient to protect against sudden onslaught. The
posterior side eyes which are second in size, guard the spider at
the sides and back. ‘Thus the entire eye-group covers about 253°
of the horizon and leaves unprotected a space on top and at the
back.

In Heteropoda (Fig. 3) the largest eyes are the posterior side eyes
The four front eyes cover a field of 145°. Between them and the
posterior side eyes there is an unprotected area similar to that in
Lycosa, of about 10°. Or since an adult of Heteropoda covers
with extended legs about 8 to 10 cm. in each direction, a spider
of the same species approaching it within this unprotected
area, would become visible at a distance of about 40 cm. The
sides are therefore very well guarded especially when we consider
that the largest eyes are used in their protection. ‘The eyes of the
front row together with the posterior side eyes cover with the inter-
ruption mentioned, fully 267°. The dorsal surface of this spider
is extraordinarily well protected as compared with the two pre-
ceding spiders. There remains an unprotected field of about

The Sense of Sight in Spiders 295

10° in front of the posterior middle eyes and about 55° of unpro-
tected field at the back behind these same eyes. In the projection
on the plane of symmetry (Fig. 7) the eyes cover .152° or in the
normal position of the spider, on a wall, 125°, as may be readily
understood from the drawing. In the vertical plane (Fig. 8) the
eyes of Heteropoda cover a field of 193°. This spider lives in build-
ings where it runs along the walls and ceilings hunting insects and
other spiders and it is distinctly crablike in motion. The com-
paratively large fields of vision in this species are possibly to be
accounted for in connection with the habit of the spider to remain
quiet during the day and to begin its activity at dusk. But this
does not obscure the fact that the sides of this spider are better
protected than the front.

It was next necessary to ascertain whether or not the fields of
vision vary in spiders of the same species at different ages. With
this object in view many spiders were examined, always with the
same result, 7. ¢., from the time when the eyes assume their
permanent position on the head of the spiderling, the maximal
angles of vision and the fields covered by these eyes are the same
as in the mature female. As to spiderlings taken directly from
the cocoon, I am sorry to say that I was unable to make any
observations upon them. ‘They are so small and their chitin so
soft that it is impossible to prepare them in the manner described
and I have not as yet devised another method. But assuming
that their maximal angles of vision are the same, which is indeed
very probable, we may readily see the advantage in the changes of
direction in the eye-axes as I have described them. A glance at
the accompanying drawings will make this clear. ‘Vo attain their
permanent position the axes of the AME in Phidippus move
upward and inward. This slight upward change makes it possible
for an image to be formed of an object on the central part of the
retina of an adult on the same plane, a good deal farther away
than is possible in the eye of the spiderling. We shall see farther
on that the central part of the retina is much more sensitive than
the periphery. The change inward tends to the same end as the
change upward and the final position of the anterior middle eyes
in Phidippus allows therefore of a more perfect distinction of

296 Alexander Petrunkevitch

objects in front of the spider. In the same eyes in Heteropoda
the direction of the axes changes in the opposite sense, 1. ¢., out-
ward and this change serves to bring about a better discernment
of objects considerably at the side of the median line, while the
same eyes still guard the front sufficiently. In the posterior
middle eyes of Lycosa, the most sensitive ones in this spider, the
direction of the axes changes to one more downward and inward
thus serving to protect better the front. The direction of the
axes in the posterior side eyes of the same spider changes in such
a way that the adult eyes look farther backward. In both PME
and PSE this change takes place at the expense of the field
protected in the young spiderling, which now becomes relatively
exposed. And here the advantage of a slower growth of the eye-
area as compared with the growth of the rest of the cephalothorax,
becomes evident. Indeed if the eye-group should occupy in the
adult spider relatively the same portion of the cephalothorax as
it does in the youngest spiderling, the unprotected field would
become in consequence of the change in the direction of the axes,
so large that the presence within it of an object even larger than a
spider of the same species, would remain entirely unnoticed. In
Heteropoda the change in the direction of the axes of the PME
is in exactly the opposite sense to that in the same eyes in Lycosa
and affords more protection to the dorsal surface. At the same
time in the axes of the PSE in Heteropoda, the change of direction
is in the same sense as in Lycosa and Phidippus. But this change
is considerably more marked in Heteropoda with the result that
in the adult spider the eyes cover fully 267 degrees instead of the
(probable) 166° in the spiderling. This idycncape cannot be
gained however, without the formation of an unprotected field.
Again, as in Lycosa, this field would have been much larger but
for the difference in growth between the eye-group and _ the
cephalothorax.

THE LIMIT OF VISION

If we examine under small magnifying power at once all eight
eyes of a cephalothorax, freshly prepared and suspended on a
drop of glycerin-albumen as I have described, we shall remark

The Sense of Sight In Spiders 297

that the four pairs of eyes form four pairs of images differing from
each other in size. As a rule we shall find that the largest eyes
form the largest images. The question at once occurs, are all
eyes equally sensitive notwithstanding that they form images
differing in size, or are the larger eyes more sensitive than the
smaller ones? The surest way to find the answer to this question
is to determine the minimum angle of vision for each eye. But
how are we to do this when we do not even know with sufhicient
exactitude the distance at which under normal conditions a spider
recognizes another of the same species. ‘The experiments of the
Peckhams, in spite of their ingenuity, still admit of too great
range for error, to be utilized in a study of the normal auele of
vision. Of what advantage, then, would be a similar experiment
but with some of the eyes ailectencd with paint? It could serve
merely to control another method, a method of comparative
morpholog gy. We have to start from the proposition that the
physiology of the nervous system is analogous in the other animals
and man, a proposition which few are disposed to admit, but here
the experience gathered in many fields and from observations
made on different animals, comes to our aid, an experience that
leaves scarcely any room for doubt that the stimulation of a single
nerve-ending transmits to the central nervous system a single
sensation only, whether or not the stimulus itself is a simple one
or in reality composed of many contemporaneous stimuli. Thus,
as 1s well known, in order to perceive two pin-pricks as two dis-
tinct sensations, it is necessary that they should be applied to
two separate nerve-endings as otherwise the sensation 1s that of a
single prick and again, when the i image of two stars falls on only
one cone of the retina of the unaided human eye, the eye perceives
but a single star. These are well known facts w hich justify us
in saying that in the spider’s eye two rods must be stimulated by
light rays in order that the image of two points should be produced.
But here the analogy ends. How strong the effect produced and
whether the corresponding image in the brain is of the same kind
as in man, we cannot know. We cannot know whether a spider
sees colors as we do, whether green appears to it in the same way
as it does to us, although we do know from the experiments of

298 Alexander Petrunkevitch

the Peckhams that spiders are able to discriminate between
colors. Neither can we know whether gradations of light and
shade are the same for the spider as for us nor how great the
amplitude of the light wave, which would be required to produce
the same effects as in us.

Nevertheless we do know that an image is formed in each spider
eye; we do know that the four pairs of images differ from each
other in size; we do know that the more rods covered by the image
the more detail can be perceived by the eye. We may thus work
on a fairly safe basis.

Let us first examine the images as they appear under the
microscope. When a black square 1s placed under the microscope
so that the axis of the eye is perpendicular to the center of the
square, it is not possible to detect any spherical aberration by
common means. But if we place the eye so that the black square
lies considerably to one side of the eye-axis, the aberration at
once becomes appreciable. For the accompanying drawing (Fig.
11) an eye of Lycosa was put so that it was a little outside the center
of the microscopic field and the axis of the eye formed a more or less
sharp angle with the slide, while the black square, each side of
which was 5 cm. long but having one side prolonged into a straight
black line, the whole made with chinese ink on a plate of milk-
white glass, was absolutely parallel to the slide. The Zeiss draw-
ing table was carefully arranged beforehand so that a small square
placed under the microscope and drawn with the aid of the Abbe
apparatus, gave on paper a perfect square. Then drawings of
the image in the spider’s eye of the black square were made from
different positions of the eye, obtained by revolving the table of
the microscope on its axis. It 1s clear from the drawing that in this
case the base and one side of the square are especially distorted.
Is the spider’s vision then distorted of all things that lie out of the
axis of the eye? It is impossible to know but I believe that the
spider forms a true idea of objects, first because the distortion in
each eye of a pair is in the opposite sense to that in the other eye of
the same pair, thus offsetting it, and second, because the retina is
not a plane but is of very complicated form differing in different
eyes and for different species. Generally speaking we may compare

The Sense of Sight in Spiders 299

the retina to a boat or canoe in some eyes and to a deep bag in
others. This may be ascertained not only from sections. It is
sometimes possible to remove the entire retina intact from the vitre-
ous body whose proximal end fills it out and to examine it in toto
under the microscope. The vitreous body also varies in shape
in different eyes and is usually considerably elongated in the
direction of the eye-axis in those eyes which form the largest
images. The vitreous body is especially long in the anterior
middle eyes of jumping spiders, Phidippus tripunctatus, for
example, and is shaped something like a long cone with its base
which is concave, toward the lens, its axis being at the same time
the axis of a conical hole which extends through its entire length.
This hole also is largest at the lens and much smaller jat the
retina and may be seen in sagittal and cross-sections. It is also
sometimes visible in young spiderlings of the jumpers, where it
presents a likeness to a pupilla. In life it is probably filled out
with a liquid.

Roughly speaking the size of the image is in direct proportion
to the size of the eye but measurements on discrepancies which
must be due to differences in the curvatures of the lenses. The
following table illustrates this.

TABLE Ix
Ratios of diameters of eyes and of images to the diameter of the AME and its image. Compare with
Fig. 10
| AME ASE PME PSE

{ ey
Phidippus tripunctatus........... ¢ BG : | 9-595 orree Sone

\ image I | 0.4285 0.0800 0.3143
Heteropoda venatoria........ steal he : | i are 1-55

\ image I | 1.75 1.50 2.00

( a e
Tey cosainidicOlaisse cc-.2)= eis .5 ale srs}s sis Q 5 ; S27 See oe

\ image I °.8 2.266 1.8666

}

This table shows that while there is a dependence of the size
of the image upon the size of the eye, this dependence is not of
such a kind as to allow of definite conclusions in regard to the
smallest angle of vision from measurements of the eyes and

300 Alexander Petrunkevitch

images in one individual and of the elements of the retina in
another. I have therefore applied two other methods. The one
consists of choosing two individuals of the same species, having
eyes of the same diameter and preparing one for the study of the
image, the other for sections through the retina. This is possible
and yields good results when one has a large quantity of living
spiders. By far the better method, the one which I now use
exclusively and upon which the conclusions I[ have reached in this
research are based, consists in preparing the spider in such a way
as to obtain from the same individual at the same time the lenses
intact for the study of the size of the image and the retina for
sectioning. This is perfectly feasible even in young spiderlings
although it requires not a little patience and experience. I pro-
ceed in the following manner: a spider is killed by a cut across
the middle of the cephalothorax so as to allow the fixing liquid to
penetrate as rapidly as possible. ‘Thus far I have obtained the
best results for the purpose with the picro-formalin mixture of
Bouin and my sublimate modification of the Gilson liquid. The
cephalothorax is allowed to remain for six hours in the fixing
liquid and is then transferred in the usual manner to 70 per cent
alcohol. ‘The sternum and the mouth parts are now carefully
removed with a fine pair of scissors. ‘Then the cephalothorax is
placed in a low dish containing alcohol and held by the side with
forceps while a thin and flexible spatula is carefully introduced
between hypoderm and chitin. Pushing the spatula slowly for-
ward it is possible to separate the entire chitinous part of the
cephalothorax from the underlying hypoderm with all its muscles
and organs. The vitreous bodies of the eyes remain with the
retinas attached by the optic nerves. ‘These are now separated
from the remaining organs, carefully noted to exclude error and
placed in separate dishes in parafhn in the usual manner. They
are then sectioned with a microtom, either parallel to the eye-axis
or perpendicular to it. ‘The sections are depigmented in chlorine
gas dissolved in 70 per cent alcohol and after washing stained in
Heidenhein’s haematoxylin. The eye-lenses of the same individ-
ual, which have been removed together with the tergum, in the
manner described, are now placed in water and carefully cleaned

The Sense of Sight in Spiders 301

with the brush on both sides. ‘They are then cut separately out
of the cephalothorax and suspended, each first on a drop of water
and then on one of glycerin-albumen and the image which each
forms of a 10 cm. square at a distance of 10 cm. is measured
with the aid of the ocular micrometer. “These measurements
may then be directly compared with those obtained from the
sections through the retina. In measuring the distance between
the rods I use the highest power only and count how many
microns are occupied by ten rods. This is essential since
the distances between the rods are apt to vary alittle. Besides, the

TABLE X

An adult Lycosa nidicola, small individual. Average distance between the centers of the two rods in

micromillimeters

Toward the At the
In the center : P a
periphery periphery
INN Up sSoooonddoodo oonbudedeauane secnudc 8 12 | 15
TAS Eyer eet tarata neice Neo cr easetartane atane SieyHine 6 10 | 12
1D No So conpescoade bdenbobognaacarooeder 8 12 | 15
1D oncaucacdoccodo gdp copDuOboOmoOUnON 9 16 21

rods are larger and farther apart at the periphery, gradually becom-
ing smaller and lying closer together toward the center. I do not
give a drawing of this but Table X affords sufficient demonstration.

It seems to me, in view of the strange shape of the retina, that
we may form an idea of the acuity of vision in the spider’s eye
only from images that cover the central part of the retina alone.
In order to diminish the possible error, | measured the image of
10 cm. as mentioned, but divided the result by ten so as to find
the size of the image of 1 cm. from 10 cm. distance.

In this way we obtain the following table:

TABLE XI
Phidippus tripunctatus

AME ASE PME PSE

Size of the image of 1 cm. at 10 cm. distance. Eye
suspended on glycerin-albumen...............--. Le 115 49 10 36

Distances between the centers of rods in the center |

Ofithe retinal. cee rtatarctss<ictejae/oislaleietals eheteraiata'alalstaeie leaden tll

302 Alexander Petrunkevitch

On dividing the size of the image by the distance between the
rods, we find that the image of a line 1 cm. long (measured in an
eye suspended on a glycerin-albumen drop) occupies respectively
38 rods in AME, 8 rods in ASE, 2 rods in the PME and 7 rods
in the PSE. One square centimeter at a distance of 10 cm. gives
an image that occupies 38? = 1444 rods in the AME, 8? = 64
rods in the ASE, 2? = 4 rods in the PME and 7? = 49 rods in
PSE. With other words the front middle eyes in Phidippus are
by far the most sensitive, then come the front side eyes, then the
eyes of the third row and last, the posterior middle eyes.

In Lycosa nidicola the difference in the sizes of the eyes is less
than in Phidippus and consequently the difference in sensitive-
ness or power of distinction is less. | examined several specimens.
Since in the one used for the measurements which I give here the
image of 10 cm. at 10 cm. distance did not coincide with the
divisions on the scale on my ocular micrometer, it was necessary,
in order to avoid error, to arrange the experiment somewhat
differently. The bristol board containing the centimeter scale
was brought nearer to or farther from the eye until a number of
centimeters not fewer than five, came to occupy a certain number
of divisions on the scale. Thus I found that at a distance of 3
cm. the image in the PME of 6 cm. occupies exactly 5 digiiene
of the micrometer scale. Each division being equal to 0.0164
mm., it follows that the image of 1 cm. would occupy 0.0164 x 2
= 0.0136 mm. Since the distance between the centers of the
rods in the center of the retina in this specimen equals 13,, it
follows that 1 sq. cm. placed at a distance of 37 cm. from the PME
will form an image occupying 4 rods (2 x 2). The image of 7
cm. placed at a distance of 36 cm. from the posterior side eye,
occupies 5 divisions of the scale or the image of 1 sq. cm. equals
0.0117? mm. and occupies 4 rods, the distance between the centers
of these being 124; but this image does not occupy the rods com-
pletely. The image of g cm. placed at a distance of 30 cm. from
the AME, occupies five divisions of the scale or the image of 1
sq. cm. at 30 cm. distance equals 0.009? mm. The distance
between the centers of the rods in this eye is gy, 7. e., the image
occupies 4 rods (2 x 2).

The Sense of Sight in Spiders 303

The image of 14 cm. placed at a distance of 27.5 cm. from the
ASE corresponds to five divisions of the scale or the image of 1
sq. cm. = 0.0067 mm. The distance between the centers of
the rods in this eye being equal to 64, the image will occupy 4
rods (2 X 2). We have then in Lycosa also, fone pairs of eyes
of different sensitiveness in the following sequence from more to
less sensitiveness: PME, PSE, AME, ASE.

Unfortunately I have no material of Heteropoda venatoria
fixed in the manner described, but from comparison with Lycosa
and Phidippus we should expect to find that in this spider the
most sensitive eyes are the posterior side eyes followed by the ASE,
PME and AME.

In order to form a clear conception of the acuity of vision of
the spider’s eye, we shall compare the anterior middle eye of
Phidippus and the posterior middle eye of Lycosa, the two most
sensitive eyes, with the average humaneye. The distance between
the centers of the cones in ae yellow spot ‘of a human retina is,
according to measurements made by various scientists, somewhere
between 4 and 5. One square centimeter placed at a distance
of 30 cm. occupies somewhere in the neighborhood of 114? =
12996 cones in the human eye, 13? = 169 rods in the AME of
Phidippus and only 2? = 4 rods in the PME of Lycosa (Fig.
12). The difference becomes still more tangible if we find the dis-
tance at which 1 sq. cm. will fall on one rod in the spider’s eye,
with other words, if we ascertain the smallest angle or limit of vision.
This may be done either by direct calculation from the data obtained
or by measuring the image of a centimeter scale that 1s gradually
moved away from the eye until the image becomes smaller than
the distance between the rods. In this way I found that the
minimal angle of vision,- using glycerin-ablumen for suspension
of the eye, equals 8’ for the AME of Phidippus and about 60’
for the PME of Lycosa, while from observation of the double
stars it is known that the smallest angle of vision in man equals
1’. Thus a creeping insect about 1 sq. cm. in size would be per-
fectly visible to the human eye, even perhaps to the extent of
recognizing the species, at a distance of about 3 m., while it would
appear merely as an indefinite, tiny moving speck to Phidippus
and would be entirely beyond the range of vision of Lycosa.

304 Alexander Petrunkevitch

These figures certainly show the great superiority of the human
eye over even the best eye of spiders. This is especially manifest
from the fact that even if we use a drop of Canada balsam with
its 1.535 refraction-coefficient, for the suspension of the eye we
still find the limit of vision for the AME of Phidippus to be 6’
and for the PME of Lycosa to be 45’... At the same time we must
not forget that the minimal angle of vision in the spiders is in
inverse proportion to the maximal angle and that with the use
of the Canada balsam the measurements would show a reduction
in the fields of vision from 40° in the AME of Phidippus to about
only 30°. On the other hand the measurements made with the
use of glycerin-albumen show that a female jumping spider
placed at a distance of 30 cm. from its mate, would still give a
sharp image covering a sufficient number of rods in the eye of the
male to be recognized by him—a result which stands in con-
formity to the experiments of the Peckhams. Perhaps this
relation between field of vision and acuity of vision was responsible
for the fact that the eyes of spiders did not attain the acuity of
vision of the human eye.

We have already seen that the smaller the diameter of a spider’s
eye, the smaller the image it forms. We have also seen that this
does not apply with exactness to different eyes of the same eye-
group but measurements show that for the same eyes in spiders
at different ages the ratio holds good as far as it is possible to
ascertain with the methods used. The younger the spider the
smaller the images in its eyes and the question arises: are the
eyes of spiderlings as sensitive as those of adults or does the power
of distinction grow with increasing age? Spiderlings that are
ready to leave the mother can be prepared as I prepared the
adult spiders and image and retina may be studied in the very
same eye. But this cannot be done with spiderlings taken out
of the cocoon so we may base our conclusions on the admission
only, that the ratio between diameter of eye and image holds
good for these spiderlings also. ‘This is indeed probable since
I have proved that such a ratio exists in all ages beginning with
the oldest spiderlings. In every case I examined for comparison
purposely the mother spider with her young ones, to avoid errors

The Sense of Sight in Spiders 305

arising from possible variations hereditarily fixed through several
generations. I scarcely need to add that several mothers and many
young ones of the same species were examined.

The distance between the centers of the rods in all eyes of the
spiderling is smaller than the corresponding distance in the eyes
of the adult. Thus in the PME of an adult Lycosa nidicola the
distance between the rods in the center of the retina was found to
be 8» while the corresponding distance in the PME of the young
is only 2.54. The diameters of these eyes are respectively 6891
and 148. Or the proportion between the diameters is $33 =
4.65, while the proportion between the rods is only = 3.2. With
other words, the image of the same object will cover a smaller
number of rods in the eye of the spiderling thanin that of the
adult, say 6 x 6 = 36 rods in the former and g X 9 = 81 rods
in the latter. The same proportion holds for all eyes of Lycosa
but when I compared the images in the eyes of this adult female
with those of her young ones that had been allowed to remain on
her back for several days longer and were beginning to leave her,
I found that this ratio already falls to 4 : 3.5 and becomes I : 1
after the next moulting. Pardosa nigropalpis showed the same
conditions and we should expect the same to be true of other
spiders, too.

CONCLUSIONS

In drawing conclusions from the facts described, I am well
aware that the methods employed in this research are far from
perfect. It is possible that the angles of the eye-axes will with
time be measured more exactly, that the figures given for the
maximal angles of vision will be a little altered in the one or the
other sense, that the minimal angle or limit of vision will be found
to be one minute larger or smaller, but in one respect the method
employed gives facts that cannot be disputed; I mean that the
error, whether large or small, is the same for the entire series of
the same kind, so that the relation or ratio will remain unchanged.
We cannot be sure that the maximum angle of vision in the AME
of Phidippus is exactly 40° or that its smallest angle of vision is
precisely 8’, but we may be sure that the larger eyes of an individual

306 Alexander Petrunkevitch

are more sensitive than the smaller ones of the same individual;
we may be sure that the eyes of the young spiderling are less
sensitive than the same eyes in the adult; we may be sure of the
proportional sensitiveness of the eyes; we may be sure that changes
take place in the directions of the eye-axes and of the direction
of these changes as well as of the time when they take place; and
all this goes to show unmistakably the existence of a perfect
balance between function and structure in the eyes as well as a
remarkable degree and an extraordinary rapidity of adaptation.
Thus when the spiderling first begins to lead an independent life,
it finds in its eyes an organ already sufficiently perfect to be relied
upon in the struggle for existence.

The changes in the angles of the eye-axes may well be looked
upon as an adaptation. I have also tried to show that the slower
growth of the eye-group as compared with that of the cephalo-
thorax, is tobe considered advantageous. “The way in which these
changes came about is also well understandable but when we ask
ourselves in what way in the phylogeny was the increasing acuity
of vision accomplished, we have to depend for the answer upon
theoretical considerations. We know that in the individual this
increasing acuity of vision is brought about as a consequence of
the slower growth of the retina elements compared with that of
the lens. Is it then a retarding influence of which nature took
advantage in order to accomplish its own end, that was responsible
or was it a possible capacity on the part of the lens to grow more
rapidly than the retina? Or were perhaps both factors at work ?
And at what time in the phylogenetic development did the eyes
differentiate into smaller and larger and why did they not all
reach the same degree of perfection? What was it that arrested
the progress toward perfection or is further perfection possible
and still in process of attainment? All these are questions before
which we stand without answer as before a door behind which
treasures lie concealed. ‘To find the key to that door would mean
to understand adaptation. For a long time science remained
satished with the explanation afforded by the principle of selection
and the attempt was made to apply this principle to all the phe-
nomena of organized life. But the very ease with which it answers

The Sense of Sight in Spiders 307

the most intricate questions proves to be its weak point and it no
longer suffices. How indeed, for example, can we accept the
explanation given for the existence of the black shield at the base
of the bill of the white swan, as a protection against the blinding
light reflected from the surface of the water, when another water
bird of the same fauna—Fulica atra—has black plumage and a
snow white shield? The difficulty is gotten round by explaining
the latter case from the point of view of sexual selection or in
some other way. The number of similar cases could be multi-
plied indefinitely. It is clear that if we desire to hold to the
principle it will be necessary to show step by step the progress
of adaptation in indisputable cases. Of this nature are variations
of which the animal itself can immediately make use to its own
advantage over its rivals. While the origin of the variations
themselves is a field for other research [ hope to have shown in
the present study of the sense of sight in spiders, the probable
course of adaptation in the survival of the fittest.

Short Hills, New Jersey
August 11, 1907

308 Alexander Petrunkevitch

BIBLIOGRAPHY

BertKau, P.—Ueber die Augen und ein als Gehérorgan gedeutetes Organ der
Spinnen. in: SB. Niederrh. Ges., 1885.

Beitrage zur Kenntniss der Sinnesorgane der Spinnen. I. Die Augen der
Spinnen. in: Arch, mikr. Anat., v. 27, 1886.

Brants—Observations sur les yeux des animaux articulés. in: Ann. Sc. Nat.,
2e Sér.,iv. g, 1838.

Coe, Leon J.—An experimental study of the image-forming powers of various
types of eyes. in: Proc. Am. Ac. Arts and Sc., v. 42, 1907.

Dani, F.—Ueber die Horhaare bei den Arachniden. in: Zool. Anz., v. 6, 1883.

Das Gehor und Geruchsorgan der Spinnen. in: Arch. mikr. Anat., v.
24, 1884.

Dujyarpin—MeEmoire sur les yeux simples. in: Ann. Sc. Nat., 5e Sér., v. 7, 1866.

Exner, Sicm.—Die Physiologie der facettirten Augen von Krebsen und Insecten.
Leipzig and Wien, 1891.

Graser, V.—Ueber das unicorneale Tracheaten- und speciell das Arachniden-
und Myriopoden-Auge. in: Arch. mikr. Anat., v. 17, 1879.

GrenacHER, H.—Untersuchungen iiber das Sehorgan der Arthropoden, insbeson-
dere der Spinnen, Insecten und Crustaceen. in: Gottingen, 1879.

HentscHeEL, E—Beitrage zur Kenntniss der Spinnenaugen. in: Zool, Jahrb.
(Anat.), v. 12, 1899.

Hesse, P.—Untersuchungen iiber die Organe der Lichtempfindung bei niederen
Thieren, vii. Von den Arthropoden-Augen. in: Zeitschr. Wiss.
Zool., v. 70, 1901.

KenNnEL, J. von—Die Ableitung der sog. einfachen Augen der Arthropoden
namlich der Stemmata der Insectenlaryen, Spinnen, Scorpioniden,
etc., von den Augen der Anneliden. in: SB. Naturf. Ges. Dorpat.,
v. 8, 1888.

KisHinouyE, K.—The lateral eyes of spiders. in: Zool. Anz., v. 14, 1891.

On the lateral eyes of spiders. in: J. Coll. Sc. Japan, v. §, 1892.

KorscueE Lt, E. unp Hemer, K.—Lehrbuch der vergleichenden Entwicklungsge-
schichte der wirbellosen Thiere. Specieller Theil., Jena, 1890.

Mark, E. L.—Simple eyes in Arthropods. in: Bull. Mus. Comp. Zoél., Harvard
Coll., v. 13, 1887.

Mutter, J.—Siir les yeux des Insectes, des Arachnides et des Crustacées. in:
Ann. Sc. Nat., te Sér., 1829.

Pecknam, G. W. anp E. G.—Sense of sight in spiders. in: Tr. Wisconsin Ac.,
v. 10, 1894.

Pryroureau, A.—Le sens de la vue chez les Arthropodes. in: Rey. Sci. Nat.
Ouest, 1891.

T he ee of Sight in S piders 309

Prateau, F.—Recherches expérimentales sur la vision chez les Arthropodes.
Pt. 2. Vision chez les Arachnides. in: Bull. Ac. Belg., v. 5, 1888.

Pritcuetr, ANNiE H.—Observations on hearing and smell in spiders. in: Amer.
Natural, v. 38, 1905.

Rases—Entwicklung unserer Kenntniss des Spinnenauges. in: Naturw. Wochen-
schr., v. 15, 1901.

ScuimKEwitscu, W.—Sur un organe des sens des Araignées. in: Zool. Anz., v.8,
1885.

Sreranowsk1, M.—La disposition histologique du pigment dans les yeux des
Arthropodes sous |’influence de la lumiére directe et de l’obscurité
complete. in: Rec. Zool. Suisse, v. 5, 1892.

Szczawinska, V.—Contribution a |’étude des yeux de quelques Crustacés et
recherches sur le mouvement du pigment granuleux et des cellules
pigmentaires sous |’influence de la lumiére et de l’obscurité dans
les yeux des Crustacés et des Arachnides. in: Arch. Biol., v. 10,
1891.

Wacner, W.—Des poiles nommés auditifs chezles Araignées. in: Bull. Soc. Nat.,
Moscow, 1888.

Watase, S.—On the morphology of compound eyes of Arthropods. in: Stud. Biol.
Lab., Johns Hopkins Univ., Baltimore, v. 4, 1890.

EXPLANATION OF THE DIAGRAMS

In the Figs. 1, 2, 3, 6 and 8 the axes of the eyes are represented by dotted lines, the maximal angles
of vision and the plane of symmetry by common black lines. In the Figs. 4, 5 and 7 the heavy black
line represents the horizontal plane for the upper part of the figures and the plane of symmetry for the
lower part of the same figures. The axes of the eyes and the maximal angles of vision in these figures
are represented by common black lines for the mature spider and by heavy dotted lines for the young
spiderling.

Prate I

Fig. 1. Phidippus tripunctatus, adult female. Projection of the eye-group on the horizontal plane
showing the axes of the eyes and the maximal angle of vision for each eye.
Fig. 2. Lycosa nidicola, adult female. Same projection as in the preceding figure.

THE SENSE OF SIGHT IN SPIDERS PLATE I

ALEXANDER PETRUNKEVITCH

Fig. 1

Tue JourRNAL or ExperRIMENTAL ZOOLOGY, VOL. V, NO. 2

Prate IL

Fig. 3. Heteropoda venatoria, adult female. Same projection as in the two preceding figures.

Fig. 4. Phidippus tripunctatus, same adult female as in Fig- 1. The upper part of the figure shows
the projection of the eye-group on the plane of symmetry while the lower part represents the projection
of the left half of the eye-group on the horizontal plane. In the same figure the axes and the maximal
angles of vision of the spiderling are indicated by heavy dotted lines and the direction in which
the change from the spiderling to the adult takes place is shown by the arrows.

THE SENSE_OF SIGHT IN SPIDERS PLATE II

ALEXANDER PETRUNKEVITCH

Fig. 3

aa ey ve y | \

Tae Journar or ExpeRiMENTAL ZoOLoGY, VOL. V, NO. 2

Prate Il

Fig. 5. Lycosa nidicola, same individual as in Fig. 2. Same projections as in Fig. 4.
Fig. 6. Phidippus tripunctatus, same individual as in Figs. 1 and 4. Projection of the eye-group
on the transverse or vertical plane. The heavy line HH’ represents the horizontal plane.

THE SENSE OF SIGHT IN SPIDERS PLATE JII

ALEXANDER PETRUNKEVITCH

SS \ vig.
Lo \ ©) Fig. 5

THE JourNnaL or ExXpeRIMENTAL ZOOLOGY, VOL. V, NO. 2

Prate IV

Fig. 7. Heteropoda venatoria, same individual as in Fig. 3. Same projections as in Fig. 4.
Fig. 8. Heteropoda venatoria, same individual. Same projection as in Fig. 6.

THE SENSE OF SIGHT IN SPIDERS

PLATE IV
ALEXANDER PETRUNKEVITCH

Fig. 7 Vee

ae
~

Fig. 8

Tue JourNnar or Exrrrimentar ZoOLoGy, VoL. Vv, No.

Pirate V

Fig. 9. Lycosa nidicola, same individual as in Figs. 2 and 5. Projection on the transverse plane.
The arrow indicates the direction in which the change in the position of the axis of the posterior side
eyes took place. The axes of the eyes in the mature spider are represented by light interrupted lines,
the maximal angles of vision by common straight lines, while the axes and the maximal angles of vision
for the spiderling are represented by heavy dotted lines.

Fig. 10. This figure shows the respective size of the image on the retina formed by a black square
placed at the same distance from each eye in the three species of hunting spiders. If compared with
Figs. 1, 2 and 3 this figure shows also that the larger eyes form the larger images.

THE SENSE OF SIGHT IN SPIDERS PLATE V

ALEXANDER PETRUNKEVITCH

Fig. 9

Fig. 10. AME ASE PME PES |G

Phidip pus
tripunctatus

(AME-1,)
SME -1
PSE -O4885

PLE —~0,08
PSE -0,3/43

Lycosa
nidicola
(AME =0,/74)

AMS - 1
ISE - 0,8
PME - 8,267
PSE - 1,867

Heteropoda

vyenatoria
(AME +026)

Aut-{
ASE-1,15
PLE 1,50
(PSE -2,00

THe Jor RNAL OF EXPERIMENTAL ZOOLOGY, VOL. V, NO. 2

Pirate VI

Fig. 11. Aberration in the spider’s eye. The figtire represents the images formed by an eye from a
perfect square which was placed in eight different places near the periphery of the field of vision. When
a square is placed in the eye-axis its image is free from aberration.

Fig. 12. This figure represents the comparative acuity of vision in man, and in two hunting
spiders. The posterior middle eyes possess the greatest acuity of vision in Lycosa nidicola, and the
anterior middle eyes possess the greatest acuity of vision in Phidippus tripunctatus. The black discs
represent a row of rods in the retina of Phidippus and Lycosa, and of cones in the yellow spot of man,
all equally magnified. The heavy black lines represent the length of the images formed by the same
o>ject placed at the same distance from each of the three eyes. In the human eye this image will occupy
57 cones, in the anterior middle eye of Phidippus nearly seven rods, while in the posterior middle eye
of Lycosa the image is only a little longer than the diameter of a rod.

THE SENSE OF SIGHT IN SPIDERS PLATE VI

ALEXANDER PETRUNKEVITCH

PHIDIPPUS LYCOSA
AME PME

Secrccccocooccsoosoccooeoos

i
a

ea
i

Tue JourNat or ExperimentAL ZOOLOGY, VOL. V, NO. 2

THE PHYSIOLOGY OF THE NERVOUS SYSTEM OF THE
RAZOR-SHELL CLAM (ENSIS DIRECTUS, CON.)

BY
GILMAN A. DREW

With One Pirate

The razor-shell clam is a particularly favorable lamellibranch
for the study of the functions of the ganglia, because: (1) It is
very active and responds rapidly to stimuli. (2) Each ganglion
supplies nerves to organs that are so active that one can hardly
fail to see movements, even when the stimulation is slight. (3)
The animal is so narrow that the shell valves can be wedged apart
enough to allow all operations and experiments to be performed
without removing the animal from its shell. (4) The ganglia
with their commissures, connectives and chief nerves, all lie so
superficially they can be seen without cutting the animal more than
to separate the fused margins of the mantle lobes and the inner
lamella of the inner gills, and to expose or cut almost any one of
them requires only the cutting of a thin outer covering that cannot
cause a mutilation that needs to be taken into account in the results
that are obtained.

Before discussing the functions of the different ganglia it 1s
desirable to study the activities of the animal as a whole and to
become acquainted with the responses of the various portions of
the body when the organs that are subject to external stimuli are
stimulated.

The habits of the animal have already been discussed in another
paper, but in studying the effect of stimuli it is necessary to know
something of the normal life of the animal, and accordingly a
brief statement of its habits are desirable here. ‘The animals are
best known on mud-flats that are exposed at low tide, but they are

1The habits and movements of the Razor-shell Clam, Ensis directus,Con. Biol. Bul., vol. xii,
no. 3, 1907.

Tue Journat or ExPeRIMENTAL ZOOLOGY, VOL. V, NO. 3

312 Gilman A. Drew

known to occur at moderate depths. Dr. K. Kishinouye writes
me that the Japanese “fishermen catch razor-shell clams from the
bottom of the sea, ten or more fathoms in depth, by means of
slender spears that are weighted at their upper ends and held at
the end of a rope.’ Inasmuch as the catch is made by simply
pulling up and dropping the spear and is dependent upon acci-
dentally striking the clams, they must be fairly abundant to make
such a method profitable. “The animal lives embedded in the
mud almost perpendicularly, with the siphon end usually barely
protruding above the surface. Occasionally specimens are found,
when the mud-flat is bare, with half or more of their shells exposed,
but, judging from observations of specimens in aquaria and of
other specimens in their native mud-flats that had not been dis-
turbed and were covered with water, I am inclined to believe that
this is not a usual position, and is probably assumed as the result
of the stimulation of the heat of the sun.

When an animal is disturbed, as by jarring the mud or by
stimulating the exposed siphons, it almost instantly disappears
into the areal This is evidently its means of escape from enemies.
The burrowing is done by means of a remarkably long, active,
cylindrical foot, Figs. 1 and 2, 7, that can be protruded from the
anterior end of the shell to a distance equal to more than one-half
of the length of the shell. When fully extended the end of the
foot is swelled to form a knob that serves as an anchor for the
animal to draw itself into the mud. ‘The fact that the animal
disappears so promptly after it is disturbed indicates that the foot
is probably kept somewhat extended when the animal is at rest
in its usual position.

The margins of the mantle lobes are fused together so that four
openings into the mantle chamber are left. “Two of these are the
openings of the siphons, Fig. 1, bs and cs, the third is the opening
through which the foot is protruded, and the fourth is a small
opening about midway on the ventral margin, Fig. 1, vo. What
function is performed by the last mentioned opening is not clear.
With an expanded animal in a dish of sea-water it is easy to demon-
strate that a current of water enters this opening. ‘This is to be
expected as the opening leads into the branchial chamber, into

Nervous System of the Razor-shell Clam 313

which water is constantly passing, and there is no reason why water
should not enter this opening as well as the branchial siphon.
When the animal is embedded in the mud however, free admis-
sion of water through this opening is not to be expected. ‘The
opening is surrounded by well developed tentacles that are similar
in appearance to those around the siphons and, like them, very
sensitive to tactile stimulations. Stimulation of these tentacles
always cause the animal to close its shell and usually, this may be
the mechanical effect of suddenly closing the shell, the slight
protrusion of the foot. The foot is almost immediately retracted
into the shell again and remains retracted unless stimulation 1s
continued. When the stimulation is continued the foot is alter-
nately protruded slightly and retracted, and occasionally, when the
animal is held anterior end downward, burrowing movements are
started.

On each side of the line of fusion of the mantle lobes are very
small papilla that are probably also very sensitive to touch. The
whole region is very sensitive but whether sensation is more acute
on the papilla than on the general surface was not determined.
Posteriorly, from the ventral opening to the branchial siphon, the
fused mantle margins are very thick and muscular. Anteriorly,
to the opening through which the foot is protruded, the margins
are loosely attached by their epithelial cells. The extensive fusion
of the margins of the mantle keeps mud out of the mantle cham-
ber during burrowing, and forms a device for expelling strong
jets of water.

Around the opening through which the foot is protruded the
margins of the mantle are much enlarged to form muscular,
thin-edged scrapers or valves, Figs. 1 and 2, c, that keep mud from
being drawn or forced into the shell when the foot is withdrawn
and the shell is forced down into the mud. It will be convenient
to refer to this portion as the collar. ‘The collar is very sensitive
to touch and when stimulated is drawn tightly against the sides
of the foot. When the foot is withdrawn it turns in over the end
and so closes the shell. Strong stimulation of the collar when in
this position, causes the margins to be drawn still further in and
thus reflected into the shell.

314 Gilman A. Drew

The siphons are the most exposed, and apparently the most
sensitive to stimuli of any portion of the mantle. They are sur-
rounded by sense tentacles and, in the expanded animal, protrude
a short distance beyond the posterior end of the shell. “Tentacles
occur all over the branchial siphon and fringe its margin. The
cloacal siphon has tentacles around it and on its sides but its edge
is very thin and does not bear tentacles. When stimulated the
siphons contract and are withdrawn between the posterior bor-
ders of the shell valves. As has already been mentioned, the stimu-
lation of the siphons of a specimen that is embedded in the mud is
the signal for its disappearance. A very slight touch, such as
might be given by a drifting weed or a piece of dirt, will cause an
instant withdrawal of the siphons but may not cause the animal
to burrow. Ifthe stimulation is repeated, burrowing is quite sure
to follow promptly. When the animal is removed from the mud,
stimulation of the siphons when not long continued simply cause
their complete withdrawal and the closing of the shell with the foot
retracted. Continued stimulation, especially when accompanied
with or preceded by the stimulation of the tenacles around the
ventral mantle opening, and with the animal held with the anterior
end pointing down, cause the foot to be protruded, swelled at
the end and withdrawn in a manner similar to the movements of
burrowing. If the stimulations are continued, these movements
are usually repeated until a dozen or more complete thrusts and
withdrawals have been made.

The foot, which is also periodically exposed to external stimuli,
is likewise very sensitive. Stimulating its surface causes its with-
drawal but it is never thrown into burrowing activity as the result.
When the foot is withdrawn, the collar closes in over it, and if
stimulation has been more than slight the siphons are retracted
and the shell is closed.~

From the foregoing it will be seen that a reasonably strong stimu-
lation of any portion of the exposed animal affects it as a whole
and may cause either complete retraction into the shell and the
contraction of the muscles that close the shell, or may institute
movements that are intended for escape into the mud. The latter
movements seem never to be caused by the stimulation of either

Nervous System of the Razor-shell Clam 315

the foot or the collar, but only by stimulation, usually when in
the proper position, of the posterior or ventral mantle region.
The habits of the animal are such that these regions are most
likely to give warning of the presence of enemies.

If instead of applying reasonably strong and repeated stimuli,
such as would be caused by stroking or pricking, very light and
short stimuli are given, such as may be given by barely touching a
tentacle with the side of a needle, a different result may be obtained.
With a specimen lying in a dish of sea-water it is possible, by
repeated slight stimuli, to cause the siphons or the foot and collar
to be withdrawn without visibly affecting other portions. The
foot and collar are so intimately associated, touching each other
as they do, that it is very hard to cause the retraction of one with-
out the other, but it is possible to cause a marked change in one
without appreciably changing the other.

Before considering reactions further it is desirable to give atten-
tion to the nervous system.

The three pairs of ganglia that are usually present in lamelli-
branchs are all well developed, but there are no other definite
ganglia. ‘There seem to be a few scattered ganglion cells about
the branchial nerves and a few others in sensitive portions of the
mantle, but on the whole the nerves, commissures and connectives
are remarkably free from ganglion cells. Although small ganglia
are reported to be present on the cerebro-visceral connectives of
Solon,” a very closely related form, I find no trace of such ganglia
in Ensis, either in the dissections of mature individuals or in the
serial sections of individuals about two centimeters long.

The cerebral ganglia, Figs. 1 and 2, cg, lie directly ventral to the
anterior foot muscles and anterior to the mouth. ‘They are far
apart and are connected by a narrow commissure, Fig. 2, cc, in
which ganglion cells do not seem to be present. Each cerebral
ganglion is joined to the corresponding visceral and pedal ganglion
by connectives, Figs. 1 and 2, cve and cpc, in neither of which
are ganglion cells abundant. Posteriorly each cerebral ganglion
sends a nerve to supply the labial palps of the same side, Fig. 2,

2 Lankester’s A Treatise on Zoology, part 5, Mollusca (Pelseneer); Cambridge Natural History,
Mollusca.

370) is Gilman A. Drew

lpn. Dorsally and anteriorly a nerve is continued to the corre-
sponding anterior foot muscle, Fig. 2, ajn. Anteriorly a large
nerve that soon branches starts forward. A portion of the first
branch of this nerve bends ventrally to the margin of the mantle
lobe and is continued posteriorly as the circum-pallial nerve, Fig.
2, cpn. The remainder of this first branch is continued forward
toward the collar. The second branch from this large anterior
nerve, aan, supplies the anterior adductor muscle. It is not always
given off at exactly the same point in different specimens, and it
sometimes happens, as in the case of the specimen shown by Fig.
2, that the origins of the nerves on the two sides are not symmetrical.
The examination of serial sections and physiological experiments
both indicate that these are the only nerves that supply this large
muscle. The remainder of the large anterior nerve is eonamied
anteriorly and sends numerous Beemehes to the collar reigon of the
mantle. The nerves of the two sides are continuous in front of
the anterior adductor muscle so a complete connection between
the two cerebral ganglia is formed, just as the circum-pallial nerves
connect the cerebral and visceral ganglia of their respective sides.
It may be well to state here that, while such anatomical connec-
tions undoubtedly exist between these ganglia, repeated experi-
ments have failed to show the possibility of sending a nervous
impulse from one ganglion to another by either of these connec-
tions. Possibly neurones from the two ganglia overlap in their dis-
tribution so there may be more pple coordination between
portions that work together.

Upon cutting the inner lamellz of the inner gills where they are
joined together, and pushing them to their respective sides, the
visceral ganglia, Fig. 2, vg, are immediately seen. They lie just
anterior to the posterior adductor muscle, sometimes, as in the
specimen shown in Fig. 2, with their posterior ends overlapping the
anterior border of the muscle. ‘The visceral ganglia are closely
fused, so there is only a slight constriction between them. The
commissural fibers are distinctly visible in sections but ganglion
cells cover them entirely. As already indicated each visceral
ganglion is joined to the corresponding cerebral ganglion by a
connective, Figs. 1 and 2, cvc, that runs along the side of the body,

Nervous System of the Razor-shell Clam 27

and by a circum-pallial nerve, cp, that follows the margin of the
mantle lobe. There is no indication that sensory impulses ever
travel from one ganglion to the other through the circum-pallial
nerve. All of the cerebro-visceral association fibers seen to be
contained in the cerebro-visceral connectives. Soon after leaving
the visceral ganglion each pallial nerve gives rise to a branch that
supplies the posterior adductor muscle, Fig. 1, pan. It is then
continued posteriorly and ventrally, sends many branches to the
siphonal region and then turns anteriorly along the border of the
mantle as the circum-pallial nerve, cpm, which joins the cerebral
ganglion. What service is performed by this connection is not
clear unless it is to afford overlap for the distribution of the motor
fibers from the two ganglia to the margin of the mantle. Sensory
fibers from the siphons all seem to go to the visceral ganglia, and
from the tentacles around the ventral opening in the mantle, to
the cerebral ganglia. For the siphons this is easily determined
by cutting the pallial nerves between the nerves that supply the
siphons and the visceral ganglia, when stimulation of the siphons
causes no response, and for the tentacles around the ventral open-
ing by cutting the circum-pallial nerves between the tentacles and
the cerebral ganglia, after which stimulation of the tentacles
causes no response. If the cut is made between the siphons and
the ventral tentacles, the effect of stimulating either portion seems
entirely normal. “The motor fibers of these nerves are hard to
experiment with but it is evident that most of the mantle muscles
posterior to the ventral opening are supplied by fibers from the
visceral ganglia. Anterior to this opening, along the path of the
circum-pallial nerves, the muscles are not very well developed.
A branchial nerve, Fig. 2, bn, leaves each visceral ganglion to pass
anteriorly and laterally to the united lamellae of the corresponding
pair of gills. “The physiology of these nerves has not been studied.
Upon stimulation of the isolated visceral ganglia, slight contrac-
tions of the posterior foot muscles have been observed, but this
may have been caused by escaped current from the electrodes.
Nerves from the visceral ganglia to these muscles have not been
found. ‘The supply of nerves to the heart and the cardioinhibi-
tory action have not been studied.

318 Gilman A. Drew

The pedal ganglia lie in the foot, very near the point where its
dorsal border joins the visceral mass, Figs. 1 and 2, pg. They are
more deeply embedded than are the other ganglia, but parts of
them may sometimes be seen when the foot is pressed ventrally and
posteriorly. “lo expose them it is necessary to remove the over-
lying tissue, which includes a thin layer of muscle. Like the vis-
ceral, the pedal ganglia are closely fused and their connecting
commissure is covered by ganglion cells. The cerebro-pedal
connectives, Fig. 2, cpc, may be seen throughout their extent with-
out cutting. Like the other connectives they seem to be free from
ganglion cells. ‘he pedal ganglia supply the nerves to the foot.
From each ganglion three large nerves pass toward the end of the
foot and one or two extend ventrally. From the postero-ventral
surface of each ganglion a nerve passes posteriorly and laterally
to the side of the foot. The pedal nerves are not easily reached
without rather extensive dissection and their individual actions
have not been studied. As they are the only nerves to the foot
they must contain both motor and sensory fibers.

In stimulating the different ganglia directly, it was found that
an electric stimulation that could just be distinctly felt by the
tongue was most satisfactory as it did not cause mutilation and
the results did not give evidence of escaped current.

With all commissures and connectives intact, the stimulation of
any ganglion visibly affected the whole animal, but the relative
time of the contraction’ of different parts differed according to the
ganglion stimulated. “Thus when the visceral ganglia were stimu-
lated, the siphons responded immediately, the collar and anterior
adductor muscle later, and the foot slightly later still. This could
be noticed without the use of recording instruments and indicates
that an appreciable time is taken in transferring from one set of
fibers to another; much longer than in the higher animals. Organs
connected directly with the ganglia stimulated always responded
first and those that were stimulated through association centers
later.

The majority of the experiments performed were to determine:
(1) The organs that received nerves from each pair of ganglia.
(2) Whether each pair of ganglia individually govern the move-

Nervous System of the Razor-shell Clam 319

ments of the organs it supplies with nerves, or whether some of the
ganglion are accessory and dependent. (3) Whether all connec-
tives carry impulses in both directions. (4) How far it was possi-
ble to have impulses transferred through association centers that
would not normally be concerned with the impulses in uninjured
animals.

Stimulation of the ganglia directly and of the nerves that leave
the ganglia, and stimulation of sensory areas and the nerves that
supply the sensory areas were all used.

In discussing the various nerves that leave the ganglia, mention
has been made of the organs they supply, so it is only necessary to
summarize here. When the cerebral ganglia are separated from
the other ganglia by cutting the cerebro-pedal and cerebro-vis-
ceral connectives and the circum-pallial nerves,* stimulation of the
ganglia causes contraction of the anterior adductor muscle, the
anterior foot muscles, the collar and at least a portion of the margin
of the mantle. The functions of the nerves to the labial palps
were not determined.

When the visceral ganglia are separated from the others by
cutting the cerebro-visceral connectives and the circum-pallial
nerves, stimulation of the ganglia causes contraction of the siphons
and of at least a portion of the mantle margins, and feeble contrac-
tions of the posterior adductor muscle. Slight contractions of
the posterior foot muscles have also been observed, but as the
ganglia lie very near them and no nerves have been found enter-
ing them from these ganglia, it seems probable that the slight
observed contractions were due to escaped current. ‘The pos-
terior adductor muscle when stimlated directly did not contract
more than when the ganglia were stimulated. It seems that it
does not function much in closing the shell. Its office is satisfac-
torily filled by the thickened, united, posterior margins of the man-
tle lobes.

When the pedal ganglia are separated from the others by cut-

5 Although the circum-pallial nerves did not seem to be able to carry impulses from one ganglion to
the other, they were cut in these experiments to make sure that the ganglia were isolated. They were
cut far from the ganglia that were to be experimented upon, so the effect of their motor fibers could be
determined.

320 Gilman A. Drew

ting the cerebro-pedal connectives, stimulation of the ganglia
causes vigorous contractions of the whole foot, including the ante-
rior and posterior foot muscles. It was not determined whether
the foot muscles were affected throughout or only in part. They
become so intimately connected with the general musculature of
the foot that a complete contraction of the foot would necessarily
involve them.

The above experiments show the organs that are supplied with
motor nerves from each pair of ganglia, but they do not indicate
whether the contraction was in each case caused by stimulating
cells in the ganglia themselves or by stimulating fibers that might
be passing through them from other ganglia.

By isolating the different ganglia and stimulating sensory areas
connected with them, motor cells can be proved to be present in
the cerebral and visceral ganglia. Stimulating the sensory surfaces
was generally accomplished by stroking with the point of a seeker
or pencil. If muscular organs connected with the ganglia con-
tracted upon such stimulation, motor cells must be present in the
ganglia.

After cutting the cerebro-visceral and cerebro-pedal connectives
and the circum-pallial nerves (the latter near the siphons), strok-
ing the tentacles around the ventral mantle opening caused con-
traction of the anterior adductor muscle, and both sides of the
collar. After separating the visceral ganglia from the others by
cutting the cerebro-visceral connectives and the circum-pallial
nerves (the latter near the cerebral ganglia), stroking the siphons
apparently caused slight contractions of the posterior adductor
muscle and strong contractions of both sides of the posterior mar-
gins of the mantle. Stimulation of one of the pallial nerves elec-
trically, caused the siphons, posterior adductor and both of the
posterior mantle margins to contract. “These experiments indicate
that sensory cells end in both the cerebral and the visceral ganglia
and that stimulating these fibers causes disturbances in the motor
cells in the same ganglia that cause the contractions mentioned.

With the pedal ganglia results are not so easily obtained. When
these ganglia are separated from the others by cutting the cerebro-
pedal connectives, the foot immediately loses its rigidity, and any

Nervous System of the Raz cor-shell Clam 221

amount of stimulation of the surface of the foot, electrically, chem-
ically or mechanically results only in the contraction of muscle
fibers in the immediate vicinity of the point of stimulation. The
foot never makes a movement as a whole and will remain motion-
less for hours, probably until it dies. ‘This seems to mean either that
there are no motor cells in the ganglia or that the sensory fibers
have no endings or collaterals in the pedal ganglia but are continued
directly through these ganglia to the cerebral ganglia. I am inclined
to believe that motor cells are present in the pedal ganglia and that
the sensory fibers pass directly through them without endings or
collaterals, for the following reasons: (1) Microscopically the
ganglia show an abundance of ganglion cells and it seems more
reasonable to believe that, in such a muscular organ, they are
not all sensory, especially as the action of sensory cells so placed,
if motor are not present, would have to be referred to the cerebral
ganglia before movement could be effected. (2) When the cere-
bro-pedal connectives are cut the foot responds with contractions.
These have the character of tetanic contractions that would more
probably come from the action of disturbed nerve cells than from
the single stimulus caused by cutting motor fibers. If such move-
ments could be caused by the stimulation due to cutting fibers
only, then the cutting of the pedal nerves (below the pedal ganglia)
should cause them, but beside the single twitch caused at the
instant of cutting no movements follow this operation. (3) If
one of the cut cerebro-pedal connectives is stimulated, the foot as
a whole, both sides, responds, apparently with a complete, normal
contraction. ‘The course of the fibers in the ganglia have not been
traced, but the effect is not what we would expect if the action is
the result of the stimulation of only half of the motor fibers that go
to the foot. It is much more easily explained by supposing that
impulses have been sent to association cells which cause the motor
cells of the foot, contained in the pedal ganglia, to act. Stimula-
tion of the nerves that leave one of the pedal ganglia, after the
pedal ganglia have been removed, does not cause complete contrac-
tion of the whole foot, as it should if the ganglia themselves have
had no effect.

Whatever the arrangement, there can be no question that the

322 Gilman A. Drew

pedal ganglia are deficient in originating power, and that when the
pedal are separated from the cerebral ganglia the foot will not by
itself, or as the result of surface stimulation, execute movements
more than are to be accounted for by the local direct stimulation
of muscle fibers. “To make sure that the movements did not come
from stimulating the ganglia, they were entirely removed and still
sumulation of the surface of the foot gave exactly similar contrac-
tions.

From the foregoing experiments it would seem that both cerebral
and visceral ganglia are able to receive impulses and to direct the
movements of certain organs with which they are connected, when
they are entirely separated from the other ganglia, and that the
pedal cannot act by themselves. ‘This is somewhat surprising
but possibly the habits of the animal may account for it. Appar-
ently the cerebral ganglia are central for the nervous system.
This is indicated by their connections with the other ganglia as well
as by experiments. “They would then have charge of the special
activities of the whole animal, as well as of the special organs in
their immediate vicinity which they supply with nerves. The
visceral ganglia govern over organs that are in constant activity, .
organs that must give warning of the approach of enemies. They
must give warning to the cerebral ganglia and then be ready to
cover the retreat by closing and withdrawing the siphons and con-
tracting the posterior margins of the mantle and posterior adduc-
tor muscle. The cerebral ganglia may now take charge of the
advance with the aid of the efficient accessory pedal ganglia.
They have little more to do during periods of burrowing. Dur-
ing the life of the animal the foot is not in a position that it will
be called upon to give such an alarm very often if ever, and dur-
ing burrowing the cerebral ganglia can devote nearly their whole
attention to the process. It is desirable in directing a retreat of
this kind to have a general in charge that is in constant communica-
tion with outposts that may give information regarding the enemy.
The cerebral ganglia have such communications; the pedal gan-
glia only indirectly.

The cerebral ganglia are the only pair that are far enough apart
to allow the cutting of the connecting commissure without injuring

Nervous System of the Razor-shell Clam 323

the ganglia themselves. With these ganglia the operation Is very
simple as they are connected by a a long narrow commissure that
is distinctly visible throughout its lenge, With the commissure
cut it was found that certain activities were delayed or otherwise
interfered with. Stimulating either ganglion apparently caused
complete contraction of the foot. The same result was also
obtained when the cerebral ganglion that was not to be stimulated
was removed previous to the experiment, or, as already noted, if
both cerebro-pedal connectives were cut and the cut end of one
of them was stimulated. These experiments again show that to
cause the action of the foot it is only necessary to stimulate one
pedal ganglion, which sets up the necessary association impulses.
If both cerebral ganglia had been left connected with the pedal
or visceral ganglia, although separated from each other, it would
have been possible that stimulation of one resulted in the stimula-
tion of the other through the pedal or visceral ganglia. Similar
experiments were tried to determine the effect upon the visceral
ganglia when one cerebral was stimulated after the cerebral com-
missure had been cut. The siphons and posterior mantle margins
of both sides always contracted completely, even though the un-
stimulated cerebral ganglion was separated by cutting connectives,
or was removed. ‘The results were thus similar to those obtained
for the pedal ganglia.

With the cerebral commissure cut and the two sides of the collar
separated to guard against a possible transfer of impulses through
the anterjor pallial nerves, although no evidence could be found
that such transfer of impulses could be made when the nerves were
intact, moderate stimulation of one side of the collar caused only
the contraction of the same side of the collar with imperfect con-
tractions of the anterior adductor muscle and possibly slight con-
tractions of the anterior foot muscle of the same side, with the
usual retraction of the foot siphons, etc. Strong and continued
stimulation however caused contraction of the other side of the
collar as well. Evidently the impulses that affected the cere-
bral ganglion that has control of this side of the collar, must have
passed by way of either the pedal or the visceral ganglia. Experi-
ment indicated that the impulses can be transmitted either way but

324 Gilman A. Drew

that the response is quicker and more marked by way of the pedal
than by way of the visceral. Much the same results were obtained
by stimulating the tentacles on one side of the ventral mantle
opening after the cerebral commissure was cut, as were obtained
by stimulating one side of the collar.

Other experiments were tried to determine to what extent
impulses can be made to travel over association fibers in other than
what would seem to be the usual ways. It was found that stimu-
lating one ganglion of a pair readily affected to its fellow gan-
glion and that the disturbance could be readily passed on from
this ganglion to others provided the transfer was of a nature
that was probably usual. For example, if the right cerebro-vis-
ceral connective was cut and the right posterior pallial nerve was
stimulated, all of the organs connected directly with the visceral
ganglia on both sides responded, and, a little later, the organs con-
nected with both sides of the cerebral ganglia and the foot re-
sponded. If the left cerebro-pedal connective of the same speci-
men is also cut and the same stimulation is given, the response
of the foot is delayed slightly but not long. In the last case it
has been necessary to send impulses from the right to the left
visceral ganglion, from the left visceral ganglion to the left cere-
bral ganglion, from the left cerebral ganglion to the right cerebral
ganglion, and from this to the right pedal, which in turn must
stir the left pedal to action with it. It will be noticed that all
transfers in this experiment are in directions that may be sup-
posed to be usual, either from one ganglion to another of a pair,
or by way of warning from the visceral to the cerebral, or from the
cerebral tothe foot. These impulses are sent so readily that in
one case it was found that by stroking the siphons of a specimen
that had been operated on in the manner described, regular
burrowing movements were instituted. Stimulation of the ten-
tacles on the left side of the ventral mantle opening gave results
almost as quickly as on uninjured specimens. Here again the
impulses from one ganglion to another are in usual directions.

On other specimens, when the left cerebro-pedal connective
was cut and the left side of the collar was stimulated, the foot
responded without delay. Impulses were moving in usual direc-

Nervous System of the Razor-shell Clam 325

tions. If now the cerebral commissure is also cut and the left
side of the collar is then stimulated, or for that matter if the left
cerebral ganglion is stimulated directly, the foot responds with
convulsive contractions only after considerable delay. In some
cases no response could be obtained. In this experiment im-
pulses must be passed from the left cerebral around through the
visceral to the other cerebral and from this to the pedal before the
foot was stimulated. “The path cannot be considered usual and
the action is both delayed and modified. Itis interesting to find
that the centers are able to respond at all in this roundabout and
unusual way.

SUMMARY

1 This form is very satisfactory for experimental study of the
physiology of the nervous system because of its shape and activity,
and the ease with which its nervous system may be seen and oper-
ated upon.

2 Continued stimulation of any portion of the body will in
time have its effect on all of the ganglia.

3 Certain organs like the siphons, collar and foot, may be so
gently stimulated as to cause them to be withdrawn without dis-
turbing organs that receive their nerves from other ganglia.

4 The relation of ganglia of a pair is quite intimate. Stimu-
lating nerves connected with one causes organs connected with
both to respond promptly.

5 Association fibers by which ganglia communicate with each
other are found only in commissures and connectives. Although
the anterior pallial nerves are united so that a connection 1s formed
between the cerebral ganglia, and the circum-pallial nerves connect
the cerebral and visceral ganglia of corresponding sides, there 1s no
evidence that the ganglia are able to communicate through them.

6 Both cerebral and visceral ganglia are provided with sensory
and motor cells. The pedal ganglia are apparently dependent
upon the cerebral for initiative. When the pedal ganglia are
isolated from the others, stimulation of the surface of the foot
causes only local responses due to the direct stimulation of muscle
fibers. It would seem that the sensory neurones have neither end-

326 Gilman A. Drew

ings nor collaterals in the pedal ganglia but are continued to the
cerebral ganglia.

7 Impulses may pass in both directions through any of the
commissures and connectives.
- 8 Stimulation may cause impulses to be sent by roundabout
connections when the usual connections are destroyed, but the
stimulation must be of considerable duration and the result is
often considerably delayed.

University of Maine
January 7, 1908

EXPLANATION OF PLATE
The anatomy of Ensis directus, Con.

Fig. 1 A specimen as seen from the right side with both valves of the shell, the right lobe of the
mantle, the right labial palps and the right gills removed. Represented with the siphons and collar
extended and the foot slightly protruded. Avery common position. Drawn from the study of dissec-
tions and serial sections and enlarged to about one and one-third natural size.

Fig. 2 A specimen as seen from the ventral surface with the mantle margins cut and the shell valves
wide apart, and the foot forced posteriorly and to the right side of the animal. The inner lamellz of the
inner gills have been separated and pushed to the sides. The ganglia and nerves in this figure have
unintentionally been made alittle too large. Drawn from dissections with a few details added from the
study of serial sections. Enlarged to about one and one-half natural size.

aa anterior adductor muscle h heart
aan anterior adductor muscle nerve /p labial palp

af anterior foot muscle Ipn labial palp nerve
afn anterior foot muscle nerve m mouth

apn anterior pallial nerve
bn branchial nerve

posterior adductor muscle
posterior adductor muscle nerve

bs branchial siphon pfm posterior foot muscle

e collar pg pedal ganglion

ce cerebral commissure pn pedal nerves

cg cerebral ganglion ppn posterior pallial nerve
cpe cerebro-pedal connective r rectum
cpn circum-pallial nerve s crystalline style

cs cloacal siphon st stomach
cve cerebro-visceral connective vg visceral ganglion

f foot vm visceral mass

g gill vo ventral mantle opening

NERVOUS SYSTEM OF THE RAZOR-SHELL CLAM PLATE I

Giiman A. Drew

Tue Journar or ExperiMeNTAL ZoULoGyY, VOL. Vv, NO.

—s7~

THE INFLUENCE OF GRAFTING ON THE POLARITY
OF TUBULARIA

BY
FLORENCE PEEBLES
With Twenty-Six Ficures

The experiments made by Loeb, Driesch, Morgan, and others,
have demonstrated that by closing the oral end of a piece of the
stem of Tubularia the development of the aboral hydranth is
hastened. The same result was obtained by Morgan (’03) when
he bent long pieces in the middle, or ligatured them so that the
ccenosare of the two ends was completely separated. Morgan
and Stevens (’04) have shown further, that the formation of a
hydranth at the aboral end of a piece produces a change in that
region, so that when this hydranth is removed, the piece is more
likely than before to develop another aboral hydranth.

The object of the experiments described in this paper was
primarily to determine what influence grafting exerts upon the
polarity of Tubularia mesembryanthemum, but one experiment
led to another until the investigations extended to a study of some
of the factors of regulation.

Last spring it was my privilege, through the generosity of the
“Association for Maintaining the American Woman’s Table at
Naples,” to spend two months at the Zodlogical Station, during
which time I carried on the experiments described in the following
pages. It gives me great pleasure to express here my gratitude
to the Association, and also to Prof. Anton Dohrn, and the other
members of the staff at Naples, for the courtesies extended to me
during my stay.

In earlier experiments in grafting (00 and ’o2) two compo-
nents having the same diameter were selected, the two cut sur-
faces were applied, and held together until the ccenosarc united.
This method proved so tedious that a new one was adopted. In

Tue JouRNAL or ExPERIMENTAL ZOOLOGY, VOL. V, NO. 3

328 Florence Peebles

order to use this method one component must be slightly smaller
than the other so that one end of the smaller one may be inserted
in an end of the larger one. The two components were usually
telescoped so that they lapped about one millimeter.

I TWO LONG PIECES GRAFTED TOGETHER IN THE SAME DIRECTION

Experiment rt. The first series of experiments consisted 1n graft-
ing together two pieces from the same region of two different
individuals, so that the aboral end of one piece was inserted in the
oral end of another piece from which the hydranth had just been

NOS -s
x “

Fic. 1 Fic. 2 Fic.4 Fic. 6 Fic. 8

removed. Each piece measured about 3 cm. (Fig. 1), not includ-
ing the hydranth of the distal piece which was not removed until
the day after the graft was made. ‘The first cut (1) removed the
old hydranth and about 4 mm. of the stem from the distal com-
ponent, the second cut (2) was made through the proximal com-
ponent a short distance back of the line of union, and the last cut
(3) removed the basal end from the proximal component. ‘These
three pieces (Figs. 1 and 2) I shall call respectively 4, B and C.
Their individual behavior after this second operation will first be
considered, and then they will be compared in order to see the
relative rate of development of the hydranths.

Grafting of Tubularia 329

(4) The piece designated 4 (Fig. 2) consisted of the major
part of the distal component, with a short distal piece of the prox-
imal component grafted in the same direction, on its aboral end.
If the long and short piece (Fig. 2, 4) act as one, we should expect
a new hydranth to form first at the oral end (Fig. 2, X) and later
at the aboral end (Fig. 2, Y). If, however, the two components
retain their individuality this result would not follow, for the oral
end of the small component and the aboral end of the large com-
ponent have had a start of twenty-four hours.

Forty-seven grafts were made, the results from these are given
below in the table.

TABLE: A
Hy dranthsratexehrets later atid eon erypateyeteleraatelerst-etcheievereicte lauetstaiescyatesstele\sfolere sfoieleverststsiavets/avers 18
Hydranths ates noneylater atl idleiamclalelnccsies cies sVersiclerniets yore 4:
Hydranths simultaneously at ¥ and Y 6
My dranthsirstia tele lateratexe sete tsyefovcteteleteterareiteralatelerete ialeleveieist ersierereletevelinictelatereveisistelelst= 6
Eby dramthe\atite One ya te atarale ata alela nisletesoreierstete) <inictetahasaiataratalalet=)-\-verntaiaia\=lelsje(asvavatefejata Aske 3
pRotalmum Deron eta ltememiveetyiiere dertrtNeiollumoranvelaotestensie crake steisterainicreeneieret feria 47

From this table it is evident that the oral end of the long piece
is the region that produces the greatest number of hydranths, and
that when they form at both X and Y (Fig. 2) they usually develop
at the oral end of the long piece before they appear at the aboral
end of the small piece (Fig. 3). About one-half of the hydranths
forming at Y came entirely from the small piece, but in the reverse
direction therefore they are aboral hydranths (Fig. 3). In the
remaining one-half both components took part, the proximal row
of tentacles appearing in the long piece, and the distal row in the
short piece (Fig. 4). A large number of the grafts (almost one-
third) developed neither hydranths nor stolons at Y. Six devel-
oped new hydranths simultaneously at X and Y. Of these, four
of the aboral hydranths were composed partly of one, partly of
the other component (Fig. 4), and two developed in the small
piece only (Fig. 5). Twenty per cent of the grafts continued
development at the line of union showing however the influence
of the second operation, for the hydranths instead of taking their
usual direction emerged from the cut end (Fig. 6). In those that

330 Florence Peebles

formed double hydranths, one at the aboral end of the long piece,
and one on the short piece, the original direction of the small com-
ponent was always maintained (Fig. 7). In one graft where the
short piece developed a hydranth in the original direction (Fig.
8) it finally pushed the graft apart and emerged.

(B) Loeb (’04) has described a series of experiments on Tubu-
laria in which he claims that the polarity was reversed. His experi-
ment was as follows: A long piece cut from the stem was liga-
tured near the oral end. New hydranths developed at both ends.
After the hydranths had emerged from the perisarc he cut a piece
out of the region aboral to the ligature. [wo days after the second
operation ten pieces had formed aboral hydranths and only five
had oral hydranths. From this result he concluded that the polar-
ity was reversed. In other words the aboral end having formed
a hydranth once, after the second operation, formed one again
because the hydranth-forming material had been carried to that
end.

In my experiment the piece B (Figs. 1 and 2) corresponds in
position to the piece described by Loeb. Instead of waiting until
a hydranth had formed at each end, the piece from which it was
cut was united with another piece at its oral end and given a start
of twenty-four hours at its aboral end. Some changes must have
been going on at these two ends as some of the pieces did not
behave like corresponding pieces cut immediately from stems that
had not been grafted. The results are given in the following
table:

TABLE 2 B

Oral hydranths not followed by aboral .vi.c.ssicicwaie 20 ie cs ewes nvceeds ane smears 23
Oral hydranths followed by aboral...............2002 000 c aes Sb are 15

Oral and aboral hydranths simultaneously

Total number, Of pieces... .<sicjcec sce sivisite cisioes selec :

The majority of pieces behaved like similar pieces in the con-
trol experiment, but in a few cases (seven out of forty-five) the
development of the aboral hydranth was hastened. Not one
piece developed a hydranth first on the aboral end.

Grafting of Tubularia 331

(C) At the time of removal of the basal piece (Figs. 1 and 2)
no sign of the tentacles’ ridges was visible, and it was possible in
only a few cases to see circulation at the aboral end. It does not
follow however from this that there was no preparation for a
hydranth. It was difficult to keep these short pieces oriented,
therefore the results are meager. [ have thrown out all those
about which there was any doubt, so that the table gives a record
of only twenty pieces.

TABLE 3 C

Hydranths:at the:aboral/end first... 60... .1..2.6 cesses Seats dodo daenedson tr 8
Hy dranths;atitheoraliendprsterorpere ctetstrerslereroriereisistetere aie elaroreretete acs terecaveheysielaieleisiccisveiaier= 6
Hydranths at the aboral and oral ends simultaneously. ....... 2... -2.. 200s eeeeeeeeeee 2
INowhy dian this ereteteletersversterrtecertereiccieiatatejatietelsietetstepaylelesaietslete/sifecatefelshatotaralaterelaste\atart eve 4
otalenum beroly pieces siyatarsrersretatescrestarete ver steisvete! state sletsjeietaesstcvedatcteye oiecclehereiclarcte sists 20

On the eight pieces forming aboral hydranths first, the oral
hydranths followed very quickly, a much shorter space of time
intervening than between oral and aboral hydranths when the oral
form first. [These double ended pieces were kept until the hy-
dranths dropped off, and a second set developed. In spite of the
fact that those on the aboral end developed first, the second set
appeared on the orals ends first, the aboral forming two days
later. ‘The pieces thus returned to their original polarity.

It is hardly possible to draw conclusions from such a small
number of pieces, but the results are sufhcient to show that the
aboral hydranths after they are once developed do not exert
enough influence over the region from which they come to estab-
lish permanently a marked polarity.

In order that we may compare one series as a whole, | have made
the following table giving the relative time of formation of the
hydranths on the pieces 4, B and C. The region of the graft is
indicated in 4 by the short horizontal line. The numbers placed
beside the pieces show the order of emergence of the hydranths,
the letter S stands for a stolon. Where no letter or number appear
there was no regeneration. ‘The arrows point toward the oral end
of each piece.

332 Florence Peebles

TABLE 4

1 1 1 1 1 1 1 1 1 1

This series of grafts shows the general result obtained from all.
It will be noticed at once, that the oral hydranth on 4 forms sooner
than that on B in nearly every case. The delay must be due to
changes going on just in front of B at the line of union, for the
pieces were originally from corresponding regions of the stems.
The oral hydranth on 4 and the aboral on C appear at the same
time, the oral hydranth on B and C at the same time, and the
aboral hydranth on 4 and B at about the same time. ‘The reason
for this is obvious, for the aboral end of C had a start of twenty-four
hours and the aboral surfaces of 4 and B, and the oral ends of B
and C were exposed at the same time.

Experiment 2. A second series of experiments, somewhat sim-
ilar to those just described, was made in order to find out (1) if
the number of hydranths formed in the region of the graft would
be increased if the two components were the same length, instead
of one being much shorter than the other, and (2) to compare
the rate of development on the oral and aboral ends of 4 and C
(Fig. 9).

Two long pieces were united in the same direction, as described
in thespreceding experiment. After 24 hours the double piece was
divided by three cuts (Fig. 9, 7,2 and 3) but this time both the first
and second cuts were made through the distal component, and the

Grafting of Tubularia 333

third cut divided the proximal component in half. The piece 4,
between the first and second cuts, differed from C not only in
position, but in time of exposure of the ends. Both ends of A
were exposed at the same time, but the aboral end of C was exposed
twenty-four hours before the oral end. In this experiment the

graft is in the middle piece (Fig. 9, B).

Fic. 9 Fie. 10 Fie. 11 iG.12 Fic. 13 Fic. 14 Fic. 15 Fic. 16

Taking the piece B first as it is made of the two equal compo-
nents grafted together in the same direction (Fig. 9) and com-
paring its later behavior with that of 4 in the first experiment,
we find that the effect of the second operation is not so evident.
Twenty-four hours after the piece had been separated from 4 and
C, on 75 per cent of the pieces new hydranths were developing on
the oral ends of the basal component, and on less than 50 per cent
of the grafts oral hydranths were appearing on the distal compo-
ponents. ‘The two components rarely acted as one piece, for one
or more hydranths usually developed in the region of the graft.
In the following table the results from twenty grafts are given.
That part of the graft which came from the distal component is
designated as D, that from the proximal component as P (Fig. 9),

If we compare Table 5 with Table 1, it will be seen at once that
there is no marked difference in the number of hydranths formed

334 Florence Peebles

on each side of the line of union. A difference was observed in
the number of aboral hydranths on the distal piece, although this
is not shown in this table. Eight out of the twenty formed aboral
hydranths in this experiment where the two components were of
the same length, and in the first experiment where the proximal
component was much shorter than the distal, only five out of
twenty-seven, formed an aboral hydranth on the longer piece.

TABLE 5
My dranthsionsthecoraltendtofy7)sersiese wave o ese esstniareceie balers ere iereus eyela'erslae ore ctmmayer se eerie 9
Hydranths onithe orallend: GRP Ne sgiareisis:sie:ag a:0.910-0 0a esas stay cecetsis,frevsuete aiuyeiere ioe uslagenietere 11
Hydranths/on-aboral. end Of Deere sates nviscc sno eit vic ieyeis ot viene nie aya eevee viet ne ole clown 8
My dranthsion;aboraliend!Of 7 cis; «/ete.o.ciavesstvsera's:mia/a:s arosd/s1s:a/alclara's ovsialavols'sve\e/ousua tere Grouacaushersts 4
Motalinumber Of pieces: cerejareisys.oveieie oie via. ovis fiers vw eyeic/erele bre ales; sueins sis Sraiwle amare 20

Before combining the results from the three pieces the behavior
of pieces 4 and C (Fig. g) will be considered very briefly. These
two pieces were about the same length, but they were not taken
from the same region of the original stems, and while the oral and
aboral ends of 4 were exposed at the same time, those of C were
not. The aboral end of C had a start of twenty-four hours. In
Table 6 the results from twenty pieces are given.

TABLE 6
Orallhydranths sie .ct: <\mojs/e aa ciate a ctajate ateia ei ariseislssclaiaed acuig sian vests ee A16 Crs
Alboralllydrassthice.eretesaricvatrscetecaisiatetotitsys(aca ers os, ziaysialelels nc (ne © 2.9.2 declaw tess A 2 Cuxg
Alboral'stolons-ercepceisals ele whehors 2c (eie cys, sue 01e-6c0 vi ocerere.a- Via desral ohh ava fara/anaate oe Al Co
IN Ose Pen ELA tLON Sey lsccte shesetsy syeiactiotarsie?®,sicye vo/sle qusvoia signs ahacatGiahe dyeceisb audieinve Have Al Crx
Motalinumberotipiecesieerasietefeveicieieiaie 0 6. oie wrarsiviche ote dusre:clers/ate 2-a/stsce se,atesne eee 20

In order to compare the behavior of the three pieces Table 7
was made showing the results from twenty-one of the double
components. In these grafts the second operation followed the
first after a period of twenty-four hours.

Experiment 3. In a third series of experiments, instead of
allowing the grafts to remain twenty-four hours before the second
operation, the time was shortened, and after three to four hours,
the pieces were isolated. ‘The results are given in Table 8.

Grafting of T ubularia 335

These results show a great difference in the region of the line of
union. The number of hydranths formed there was greatly

TABLE 7

Pat

ee
LS)
=
[ian
caer 00 3
EX) o
LS)
ro)
EL) L<)
wD a
wo
[= w—3
=
\— o
i -
Ce a a
=)
=
=
> >
w

3 3 2 2

UETLEULELITLAT|

reduced, the double pieces resembling a single one in their behav-
ior. Pieces of the same length as 4 and C were cut from corre-
sponding regions of stems which had not been grafted. A com-

2 |2 |2
2

1 i 1 PRS ele Me

TABLE 8
2a | es Lm {pt
x is
2 2 2 2
1 1 1 1 r rx
B | 1
1
rn
Ss 1 1
3 3 1 1 1 2
c aA
1 4 2 il 2 i

parison of their behavior with that of these pieces cut from the
grafted stems shows a marked difference in the number of hydranths

336 Florence Peebles

developed. A much larger per cent of aboral hydranths formed
on 4 inthe control, and also more oral hydranths on C in the con-
trol. This seems to indicate that the process of hydranth forma-
tion at the grafted ends of these pieces affects their later behavior.

Table 7 and Table 8 show very definitely, that the results after
the second operation are not modified by those following the first
unless the period between the two operations is sufficiently long
for them to get a start. In the first place (Table 7) the number of
oral hydranths on the distal half of the original distal component,
is much larger than that on the proximal half, while there are
more aboral hydranths on the proximal half than there are on the
distal half. ‘This is not the case (Table 8) when the second opera-
tion follows the first after a very brief period. Secondly, the
number of aboral hydranths on the proximal end of the original
proximal component is much larger than the number on the distal
half of the same component, and the number of oral hydranths
on its distal half is greater than on the proximal half. ‘This is
not the case when the time between the first and second operations
is reduced.

Experiment 4. In the fourth series of experiments the two
components were grafted together in the way described above, but
this time the level of the second cut was changed, making the
piece 4 (Fig. 10) consist of the greater part of the distal compo-
nent while B (Fig. 10) was made up of a short basal piece of the
distal component grafted on the oral end of the proximal compo-
nent. The third cut (Fig. 10, 3) was made nearer the aboral end
of the proximal component thus making C shorter than in the
preceding experiment. A series of sixteen grafts of this descrip-
tion are represented in Table 9.

If we compare the rate of development of hydranths on 4,B, and
C we find oral hydranths on 4 and C appearing at about the same
time, also those on the oral and aboral ends of B. The per cent of
hydranths on the oral and aboral ends of 4 and C was about the
same as that in Experiments 1 and 3. The proportion of double
hydranths at the region of the graft was larger, and also the num-
ber of aboral hydranths on the distal end of the proximal compo-
nent. When this experiment is modified by decreasing the time

Grafting of Tubularia 337

between the first and second operations (Table 10) no hydranths
formed on the oral end of the proximal component in piece B, but
a large number of the distal short pieces formed hydranths. A

TABLE 9

Soa eet tame cee enaemn ea Peale a ate. teeta

1 4 b 2 |2 4 la le
ae 2 2 2 P 2 2 2 2.
fo)
Lar dite a rh bE |e op a pe IRS ree Hh

very small number of hydranths developed on the aboral ends of
A, in this series not one. The oral hydranths on A and C appeared
about the same time.

TABLE 10

i 1 1 1 1 1 1 i 1 ly
A

{1 Ss 1 A il [ *
Me + + + + | + |B

2 2 2 ‘1 2 2 1 3

eat eit in 1
o

2 1 1

338 Florence Peebles
2 TWO LONG PIECES GRAFTED TOGETHER BY THEIR ORAL ENDS

Experiment 1. Two pieces of stem, each measuring about 3
cm., were grafted together by their oral ends (Fig. 11). The
grafts were left undisturbed for twenty-four hours, then the double
piece was cut at two levels (Fig. 11, 7 and 2) so that each compo-
nent was halved. The distal halves of each were united by their
oral ends forming the piece B while the proximal halves of each
(4 and C) had their oral ends exposed by a fresh cut, their aboral
ends having a start of twenty-four hours. New oral hydranths
formed on 4 and C at practically the same time, the aboral usually
preceding the oral by a few hours, or forming simultaneously with
it. The piece B developed a very small proportion of hydranths
at its free aboral ends, but in nearly every graft double heads
formed, one on the oral end of each component; these emerged and
finally pulled apart. These oral hydranths were much slower
in developing than the oral hydranths on the free ends of 4 and C.
When the second operation followed a few hours after the first,
the percentage of hydranths formed at the line of union was greatly
reduced. ‘The free aboral ends rarely developed hydranths at the
same time, one usually preceded the other by six or eight hours.
The oral hydranths on 4 and C in this experiment formed before
the aboral hydranths with very few exceptions.

Experiment 2. In a second series of experiments in which the
two components were grafted together by their oral ends (Fig.
12), the level of the first cut was changed so that 4 consisted of the
major part of one component, while in B instead of the components
being equal in length, one was much longer than the other (Fig. 12,
A and B). A period of eighteen to twenty-four hours elapsed
between the first and second operations. Table rr gives the
results from eighteen grafts. These practically represent the en-
tire series of experiments so that it is unnecessary to give other
tables.

If we consider the rate of appearance of the hydranths we see
that the percentage of aboral hydranths is very large, and that
they appear before the oral hydranths of the same piece with few
exceptions. [he number of hydranths on the oral end of the

Grafting of Tubularia 339

large component of the graft is greatly reduced, while a relatively
large number form on the aboral ends of the smaller component.
The pieces 4 and C, upon which the aboral hydranths appeared
before the oral, were kept until the first hydranths were lost, and
new ones developed. Without exception, the second set of oral
hydranths appeared first, even in cases where the aboral hydranths
had developed one to two days earlier than the oral ones. This
shows without doubt that the polarity is not permanently reversed.

Experiment 3. In this experiment the second operation fol-
lowed the first after six hours The results show that the aboral
ends had not had a sufhcient start to produce hydranths before

TABLE 11

hy ey ee} 22S) A Les eet ale ol
a 2) 2) (2 2 (2)
c

Dy te ay ie ee re TE py be Ee Tp

the oral ends. None of the grafts formed hydranths on their oral
ends, instead, the majority developed a hydranth first at the aboral
end of the short piece, and several hours later at the aboral end of
the longer piece.

3 TWO LONG PIECES GRAFTED TOGETHER BY THEIR ABORAL
ENDS

It has been demonstrated by many observers that a piece cut
from the stem of Tubularia, if sufficiently long, shows marked
polarity, 1. e., a hydranth develops first on the oral end of the stem
and then later on the aboral end. I have shown that when two

340 Florence Peebles

long pieces are grafted together in the same direction, they may
act as one piece, a hydranth forming first at X and later at Y
(Fig. 2) but a large per cent form hydranths simultaneously at
the oral ends of each component (Fig. 4). The question naturally
arises as to the result if two pieces are grafted together by their
aboral ends so that the two oral ends are exposed.

Experiment r. ‘Two pieces each measuring about 3 cm.
with the hydranths still attached, were grafted together by their
aboral ends (Fig. 13). At the end of twenty-four hours the hy-
dranths were removed, together with 1 cm. of the stem. The
graft (4) consisted of two components of equal lengths whose
aboral ends had been united for twenty-four hours and whose oral
ends were exposed at the same moment by a fresh cut. This
experiment was repeated, with modifications, several hundred
times. ‘The results can not be combined in tables without much
repetition, therefore I shall merely give one representative series.

TABLE 12
ria bh 1 ff ft Pi. ee f(t 14 j2 j2 jd fl 2
bt 2 |2 1 2 |2 1 1 n it 1 1 aL

In this series no hydranths formed at the graft line. In most
cases the two components acted as one piece forming a hy-
dranth at one end only, or first on one end, and then on the other.
Whether stolons would have developed later, I can not say, for the
pieces were kept only four or five days. At the end of that time
although two sets of hydranths developed on some pieces there was
no sign of stolon-formation or pushing apart at the aboral ends.
That one piece is influenced by the other seems evident from the
results. Only five grafts formed hydranths on each end at the
same time, while ten produced one first at one oral end, and then
after one to two days, at the other oral end. Ten more formed
hydranths at one of the oral ends and nothing at the other.

Experiment 2. Ina second experiment one component was cut
close to the line of graft (Fig. 14,2). In this way, it was thought,

Grafting of Tubularia 341

that the long piece might exert some influence on the short one.
The results from one series of twenty-seven grafts is shown in
Table 13.

Nine of the long pieces formed hydranths on the oral ends first,
while this took place only twice in the shogt pieces. Nine of the
long pieces formed hydranths while no new hydranths appeared
on the short ones.

Experiment 3. In order to determine if the distance from the
original hydranth had any influence on the rate of development,
a set of experiments was made where the pieces were grafted so
that when cut the oral end of one component should be much
nearer the original hydranth than the oral end of the other (Fig.
15). The results showed no difference in the time of develop-
ment of the oral hydranths on the two components.

TABLE 13
fPppeP 2 |2 \2 1 2 2 2/8 2 2
Yh ah DUTT teil Tee tt oitte ne oe deta Koike BE hit a oh

Experiment 4. In the fourth experiment the pieces forming the
graft were cut off at equal lengths, very close to the line of union
(Fig. 16), each piece measuring 2 to 4 mm. With scarcely any
exceptions (about three out of forty), the hydranths formed first
on the oral end of the inner piece ( Fig. 16, X) and if one formed
at all on the outer piece (1°) it appeared at least one day later.

The results of these four series of experiments seem to me to be
of peculiar interest, and they are not without weight in consider-
ing the problem of polarity. Why should a compound (grafted)
individual with two oral ends exposed at the same time, in a large
majority of cases develop a hydranth first on one oral end, and
and then on the other? Shall we say that there is such a thing as
polarity in this new double individual, or shall we say that all the
“hydranth-forming material” has been carried to one end so
that development at the other was delayed? It is evident from
experiments that I shall describe later, that the direction of the

342 Florence Peebles

current has nothing to do with the order of appearance of the
hydranths on the ends of the grafts. We must seek an explana-
tion elsewhere. I believe that it requires a large amount of energy
to construct a new hydranth. In order to produce sufficient
energy certain metabolic processes are set up. ‘These processes
must begin as soon as the wound closes. If the condition of the
stem is such that sufficient energy can be produced, hydranths are
formed at once, if not, the development is delayed until there is
enough energy. When two pieces are grafted together, some of
this energy is expended in healing the wound, and uniting the
pieces. If there is a large enough quantity left over, or already
in the pieces, hydranths develop at once at the two oral ends, but
if there is not a large enough amount present, one end is delayed
until the hydranth has been completed. This hypothesis may
serve to explain the hastening of the aboral hydranth after one has
been formed or is about to form in that region. If the hydranth
has formed there may be energy left over, if it is about to develop
there is a large amount of energy present. Under normal condi-
tions some stems contain more energy, or vitality. The preceding
tables show that from the two original components a large number
of hydranths may develop as many as eight, while from others only
one or sometimes none, appear. ‘The conditions of the experiment
are apparently the same; the results can be explained in no other
way than that one individual possesses more energy than another.
I do not believe that there is any one material whose presence
modifies the result, it is the state of all the materials at the time
of the operation.

3. GRAFTING A SHORT DISTAL PIECE ON THE BASAL END OF THE
SAME STEM

In an earlier paper (00) I described a series of experiments in
which a short distal piece of the stem was grafted in a reverse
direction, on the proximal or aboral end of a long piece. The
results which I obtained from a small number of experiments,
seemed to indicate that the long piece influenced the rate of develop-
ment of a new hydranth on the short piece, for the tentacle ridges
on the short piece did not appear until after the hydranths had

Grafting of Tubularia 343

emerged from the oral end of the longer piece, a region which was
nearer the basal end than the short piece. “The number of experi-
ments was almost too small to warrant any definite conclusion.
I have repeated this experiment a number of times and have finally
come to the conclusion that the major component does not in-
fluence the minor one unless it shares in the formation of the

hydranth which develops at that end.

LE ht

Fic 17 Fie. 18

Experiment 1. A short piece measuring about 1.5 mm. was
cut from the distal end of a long (3 cm.) piece from a region near
the hydranth. This small piece was then grafted on the aboral
end of the long piece (Fig. 17) so that the oral end of each piece
was exposed, the aboral ends united. ‘Twenty-seven grafts are
represented in Table 14. The dotted line shows the original
position of the small piece.

TABLE 14
a ee a AT De PIO retells Meg's SU Olea aa Tate) SO Tg eo Ne a(t a

re lee he ee Ooty Bie Te ea re fT ott} seri Seer eae) aL eer)
| Tie L TAU Tete EME UTE) pies al ETE | PT TRG PE PPC Mb taTFE Mb U rk. iby

1 1
Lie le 2 le 2 2 HT 2 le be lt 2 es

Eleven of the twenty-seven grafts formed a hydranth first at
the oral end of the major component (P), and later at the oral end

344 Florence Peebles

of the minor component (D). Eleven developed oral hydranths
on the long piece and nothing on the shorter one. “T'wo developed
hydranths on the short piece only, and two on the aboral end of
the long piece only. In one case new hydranths appeared simul-
taneously on the two exposed ends. In all grafts where a long
and a short piece are united, the formation of a new hydranth 1s
always slower in the short piece. In six of the eleven grafts that
developed the hydranth first at the oral end of the major compo-
nent, the hydranth that formed later on the other end came partly
from the long piece and partly from the short one, i. e., the distal
tentacles developed in the minor component, and the proximal in
the major component. In these the development was always
slower.

4 GRAFTING A SHORT BASAL PIECE ON THE DISTAL END OF THE
SAME STEM

In order to test the influence of a long distal piece on a short
basal one, a second series of experiments was made. ‘This time
the hydranth was cut from a piece of stem measuring 3 to 4 cm.
From the basal end of this piece a short piece (2 to 3 mm.) was
cut off, and grafted in the opposite direction, on the oral end of
the same stem (Fig. 18). The results from these experiments
were not surprising, Here no influence seemed to be exerted by
the major component, as the following table shows.

TABLE 15
Waite le Peale ;; .
1 fa 1
D
1 2 i Lie 1

No very definite conclusions can be drawn from this table, or
from any of the other series in this experiment. ‘The major com-
ponent apparently took no part in the formation of the hydranth
in the smaller piece. From constant observation of the behavior
of grafts composed of a short and a long piece, I am inclined to
believe that the size of the short piece has more to do with the rate

Grafting of Tubularia 345

of regeneration than contact with the major component. The
only cases where it seems to me we are justified in looking for the
influence of one upon the other is where the hydranth is developed
partly in the long piece and partly in the short one. This did not
take place in any of the experiments.

§ THE INFLUENCE OF THE CURRENTS ON REGENERATION AFTER
GRAFTING

Experiment 1. Two short pieces grafted in the same direction.
It was suggested to me by Professor Morgan, that the current in
the two pieces of which a graft is composed, may have something
to do with the order of regeneration in the outer ends of grafts.
Soon after a piece is cut from the stem of Tubularia, the wound
closes, and rapid circulation begins. The current is easily seen
coursing up one side of the piece and down the other. When two
pieces are united the currents do not always flow from one piece
into the other. Instead, the current may be seen flowing up one
piece and turning at the line of union as if stopped by a membrane,
and continuing down the other side. Frequently, however, the
current continues to flow up one side and on into the other piece.
In Fig. 19 (E and F) these two cases are shown. In £ the current
is continuous; in F it is separate in the two pieces.

Small pieces, measuring 1.5 to 2 mm. in length, were cut from
different stems from a region at least 1 cm. back of the hydranth.
One piece was inserted in the other so that the oral end of one
overlapped the aboral end of the other (Fig. 19, 4). At the end of
twenty-four hours each graft was carefully examined under sufh-
cient magnification to detect the direction of the currents. All of
the grafts in which the circulation was continuous from one com-
ponent to the other (Fig. 19, £) were put in lot 4, while those in

346 Florence Peebles

which the circulation of one piece was distinctly separate from
that of the other (Fig. 19, Ff) were isolated in lot B. Another lot
(C) consisted of those in which the circulation was irregular, and
the last (D) in which no circulation whatever could be detected.
The rate of development of the hydranths is shown in Table 16.

TABLE 16
ae B {0} D
he 1 1 ji 1 Loni tel rib a |e)
| 2 2 2 2

The behavior of the grafts is practically the same whether the
circulation is continuous or not. In each case a hydranth devel-
oped first on one piece and then on the other or on one piece only.

Experiment 2. Two short pieces grafted together by their oral
ends. Pieces of the same length as those in the preceding experi-
ment were grafted together by their oral ends, so that the aboral
ends were exposed. They were separated as before into lots 4,
B,C and D. The results from one series are shown in Table 17-

TABLE 17
A B c D
\ fled 1 Oe iti het bie tt Use
lls 1 2 1 2 |2

Of these thirteen grafts four developed simultaneously on the
aboral ends. Three formed one first on one end, then on the |
other, five formed one on one end and none on the other, and one
developed a hydranth on one end and a stolon on the other. A
control experiment was made in which single pieces, the same length
as the entire graft, were cut. Out of eleven pieces, eight formed
a hydranth on one end and nothing on the other; two developed
a hydranth first on one end, and then on the other, and one devel-
oped a stolon on one end, and a hydranth on the other.

Experiment 3. Two short pieces grafted together by their aboral
ends. ‘These pieces were the same length, and taken from the
same region of the stem, but were grafted by their aboral ends

Grafting of Tubularia 347

(Fig. 19,C) so that the oral surfaces were exposed. A series of
twenty-four grafts is represented in Table 18.
TABLE 18
A B
if} 1 TY rT ee
{le suleche. te 2
Out of twenty-four grafts, twelve developed a hydranth first
on one end, then on the other in spite of the fact that in some the
current was continuous in the two pieces and not in others. Only
one developed hydranths simultaneously on the free ends. Eleven
formed a hydranth on one end only.
These experiments, as a whole, show that double pieces usually
form one hydranth only (Fig. 19, D), or first one and then another

later on the other end, regardless of the direction of the graft, or
the flow of the currents.

c D
Lit [Le Me (ttt te

2 2 2

6 THE EFFECT OF INTERRUPTING THE NORMAL PROCESS OF
HYDRANTH FORMATION

Driesch (’97) first showed, in his researches on Tubularia, that
when the formation of the hydranth is interrupted by separating
the two tentacle ridges shortly after they appear, the method of
completing the hydranth is not always the same. He has described
four methods of regulation: (1) The ‘‘ Regenerationsmodus”’
where the hydranth emerges with the original proximal tentacles,
and later develops a new distal row; (2) the “ Ersatzanlagemodus”’
where the coenosarce in front of the proximal row elongates and a
new distal row appears before the hydranth emerges; (3) the
“ Auftheilungsmodus”’ where the prox mal tentacles divide, form-
ing the distal row from their distal ends; (4) the “ Auflésungs-
modus” where the proximal row disappears entirely and a new
anlage forms. I repeated these experiments (’00) suggesting that
the difference in the method of completion of the hydranth on the
proximal piece was due to the degree of differentiation of the pri-
mordia. If the distal row of tentacles was removed soon after the
red material had begun to collect in the two rows, the fourth
method was invariably followed, 1. e., the first proximal row dis-

348 Florence Peebles

appeared and the complete new anlage developed. If on the
other hand the two rows were separated later after they were well
defined, the first method of completion was followed. The prox-
imal piece is, therefore, as Driesch has shown, capable of com-
pleting itself in a distal direction. The small piece (4) bearing
the distal row of tentacles, is also able to complete itself distally,
but as far as | am aware no one has found that such a piece is
capable of forming new proximal tentacles, thus completing itself
in a proximal direction.

It seemed to me worth while to repeat this experiment 1n order
to find out at what time the distal piece (Fig 20,4) becomes so
highly differentiated that it is no longer able to complete itself
proximally, and also to observe the other methods described by
Driesch, especially the “ Auftheilungsmodus” which | had never
seen, although I had repeated the experiment more than a hundred
times.

In order to find out the exact time when the small piece (Fig.
20, 4) becomes too highly differentiated to complete itself, it was
necessary to remove the tip of the stem before the tentacle ridges
were visible. Driesch tried this on forty-five pieces, allowing
about twenty-four hours to intervene between the first and second
operations. Out of these forty-five pieces, thirty-seven developed
one row of tentacles, two formed a double row, and six a complete
hydranth without a stalk. Five of the six pieces that formed a
complete hydranth were “zu gross,”’ therefore he considers that
there were only three out of forty which developed more than the
distal row. He concludes that there is therefore a definite time
before the anlage appears, when the character of the further
development of the smaller tip is determined. In order to deter-
mine the exact position of the tentacle ridges before they are visible
on the outside, it was necessary to measure a number of anlagen,
then to take the average length. Fifty pieces were cut from dif-
ferent stems, and left undisturbed until the anlage was visible on
each. Measurements were then made, first from the tip of the
stem to the base of the proximal tentacles (Fig. 21, P), and second
from the tip of the stem to the base of the distal tentacles (Fig. 21,
D) the average length of P was 1.7 mm. of D .6 mm.

Grafting of Tubularia 349

Experiment 1. Removal of the region of the distal tentacles
before the ridges appear. The hydranth and about 5 mm. of the
stem below it were removed from twenty-seven pieces of stems
whose length averaged 3.5 cm. These pieces were left undis-
turbed for eighteen hours. At the end of this time the circulation
in the oral end was rapid but no ridges had been laid down. A
piece .7 mm. long was then removed from the oral end of each
piece (Fig. 22,4), thus separating the region in which the distal
tentacles would appear later, (4) from the longer proximal piece
(B) on which the proximal row would have developed. The
pieces were isolated and their later behavior observed. ‘The
following table gives the results from twenty-five pieces, two of the
twenty-seven having been lost.

SERIES 1 A
Completes hy dram the ater eyes tetas =a) slew lene svete fw = ere 2s incl SEES Gdy erste eee cle tei te 5
Distal'tentacle som yicer. jatar sYoy-seyepetois ee) «: shat atsvats ove sis lojaretove)\etelolel nt evstehete Var eieie eretereetantsys 10
IND VEEN Cirsb) No yn cgi occocHonuedoddne Seanoonppusuehosbansespocpaneaeore cece 10
MBq tal bmataen beret 2 yot 2 -\a)o era toyet=, oYei<ysisvavasarsiata/s (alee si ejatars) so) aVei die ola ers eforhagelnictamtensictaloeeis 25

By complete hydranth I mean distal and proximal tentacles, and
reproductive organs. No stalk developed on these small pieces.

In the second series, twenty-two pieces were cut, the length of
the distal piece (4) was increased from .7 mm. to I mm., thus
including part of the region between thetwo rows of tentacles.
The time between the first and second operations was the same as
that in Series I.

SERIES 2 A
Complete hydranth 9
Lise eal Rte 695 oS bans onde nasenccoontecodoc pop Rup ASbdabbupondoDOssdcopDea sos 9
Nope genera tionlartayerapctevatsre tale iate( relator cra\etetoieyacte vols hele (oisYo a)a/elsteie\carniaiars\ ste e/alals(ersisvelerersier sas 4
eo Gall ena ran Se Ys areetete ta toner a taledeteyole tetateteneto\atarsUoreracalerelal ais, oYalela elalaislaieiatorspaictotatareleteiebetsteeiete 22

Comparing Series 1 and 2, it will be seen that with an increase
in the length of the distal piece (4) there is an increase in the pro-
portion of complete hydranths.

350 Florence Peebles

In a third series 4 measured .8 mm. This time the tip was
removed as scon as the piece was cut from the stem, so that no
time elapsed between the first and second operations.

SERIES 3 A
Complete why dranthityetstetateters oreteteteer totic cele) atnieteta/a/ebeleta ore oitiose ier elele eiele(ousroVoreeioieiiefeeren yer °
ID istalGtentacles merce cete(ats/etts stele paistePevetetalelaislaveetstsja:aiels’eieisy svat (clelenshayeye(s ciafeteYefeyntezetsbereusisyets 7
LD eT) Ge ee ee ac maa (on G ee On O CCC InO OCH eE EEE OnE ar Price aEee ne ssrida I
INowre genera ion crrarspamteteteccrete ctetepeh sleretete ra ciaTots ie cher aus avarskalatave:aicial o(a,e/e¥aeve ajsieqecateasterereiolepeterete 4
pLotallentumbex meters ciietteiete tess iohe nielei- ei Inve (3) «blo ale rie.o npn ones een eee eee 12

In this series one of the “incomplete” structures, i. e., a hy-
dranth composed of two distal rows developed. This result is
often obtained from short pieces. The piece 4 in this experi-
ment was shorter than the short pieces from which Child (’07b)
obtained a complete hydranth.

In a fourth series the anlage of the proximal row of tentacles
was just visible as a faint red area. The length of the piece
removed was .6 mm. ‘The time between the operations was forty
hours. The following table gives the results from thirty pieces.

SERIES 4 A

Completembydranthaseyerereperateteves ove oleyeteo:ets;c\cFaiehe’o:e\aietevele)ats eiaisscyeloloteisists,cksieya crs eee eo °

Distal stentaclesiseree-<meeissciie ss. ssc, « pure /nveicla apd fers oie dO) e1evece fee cei ols Lee Epa Te ME Te 2

INO} regener atin aerstire:recePaiehsosejore isis: \eisi~e!s slotoie soba /ots\arcis tr dvcveratecahs om big efstavat stants persia 5
otal enumberNerseiserte sis sisteicieve sels avsicie = sen sre Syalete’ajeieVelene (oxele talafoissaele ote e eke eteteter 30

Here no complete hydranths developed, while twenty-five
formed the distal row which was probably already laid down
when the two pieces were separated.

In a fifth series the piece 4 was removed immediately. This
time the tip cut off exactly equaled the average area of the entire
anlage (Fig. 23,2):

SERIES 5 A

Complete phy dran thee eeteteteleteisss etotsid) leiolelstelajols Zaire st storie ;

8 Saternererchaine cee eee 12
Distal litentacless-accrue sts eh carci sbefoniete latte aie otic eieare es SIE TE eee tire 3 °
INomre pen era toner eter tacre ayers rpc raatelete he ieisterace/sreiclat gps (altel ste cwors, o:61erehd saree eS 3

eo taliionumbexrctsyers 2 cyetwts evel voreleyo\ste,che kei over ess aoonneacec

Grafting of Tubularia 351

This table shows that the small piece if isolated before any of
the processes preparatory to hydranth formation are begun, 1s
not only capable of forming a complete hydranth, but in a large
proportion of cases it does form the complete structure. The
length of time between the operations and also the size of the tip
removed are factors in determining the extent of later regeneration
in the end of the piece.

The later behavior of the proximal piece (Fig. 20, B) is given
in the following parallel series where the tip was removed before
the ridges appeared.

SERIES 1 B

Old proximal row retained, new distal infront..............0 000s eee e cece eee eeeee 10
Nie wa rekangeeerc stonsyeisystoiarevarecstaotel ohatetsts Rita aren ae EPahsts hea ahagatla eee eee dee clea one eters 5 itl

Notallnum bers ees sas :

wjelle/ssis|ehs/eiaielele|ele\sheteieis)siete - 25

It will be seen upon comparing this series with Series 1 A in
each there were ten pieces that retained the original tentacles that
had been laid down; these ten pieces were parts of the same piece
before the second operation. The fifteen proximal pieces in
Series 1 B which formed entirely new primordia, developed on their
original tips five complete hydranths, while ten died without any
further development.

SERIES 2 B
Oldiproximal row retained\/new|distal\inifromt. 5525.02 wie. « wie)e oinressie siey= wieneyernlejatevaieel eee 9
INewaanl ape trrreusrsrot stofes,sieretoiaveseisis a> Se charatehohekitoheyopois) eteieis a ctarag tate vatenshekane heen 13
Motalemium berks) ac /elsrsniéiaie/s cel e/s,2 5 (ots 2 Boca pagde 22

Again it will be seen by comparing this series with Series 2 A
that some of the’ proximal and distal pieces retained the original
anlage which had been started before the second operation, the
larger number, however, developed a new anlage. Series 3 and
5 B will not be given as the pieces developed new hydranths in the
usual way. :

In the fourth series, the second operation was postponed until
after the proximal ridges were discernible, but the distal had not
yet appeared.

352 Florence Peebles

SERIES 4 B
Old proximal row retained, new distal in front. .......-..0 +e eee ee cece reece eee eees 22
IN Git Ea oucanob po ddodoobpUgnton nnd pauabUSobUeucOnGope ROBO AE DUDE On ioonds. 8
Wotalsmumber’ 2; 1a csc/sic/saysle : Se etevere ayeyete etek ete crescent ais asat ale! eiaty afeqstetensveteete 30

The method of completion of the hydranth on the proximal piece
(B) when the original row persisted, in Series 1 and 2 was that
described by Driesch as the “Ersatzanlagemodus,” a new distal
row developed in front of the proximal row before the hydranth
emerged. That in Series 4 was Driesch’s “ Regenerationsmodus”’
where the hydranth emerged first, and later a new row of distal
tentacles developed. I agree with Child (’o7c) that there 1s no
essential difference in the two methods.

CONCLUSIONS

These experiments prove that before the ridges have become
visible, changes have taken place in the tip of the stem that render
it when isolated incapable of producing a complete hydranth.
These changes however do not take place until several hours
after the wound has closed. If the tip is removed shortly after
closure of the wound, even as long as twenty-four hours after the
first operation, it is still possible for the isolated tip to form a com-
plete hydranth. After the proximal tentacle anlage has appeared
the distal piece when isolated, even if the tentacle ridges are not
visible, does not form a complete hydranth. — Instead the distal
tentacles and mouth develop.

7 REVERSING THE DISTAL ROW OF TENTACLES

In earlier experiments (’00) I have shown that when the tip
of the oral end of a long piece is cut off, reversed, and grafted back
again at once, that the new hydranth develops in exactly the same
region as it would have if the piece had not been removed. The
distal row of tentacles appears in the small piece and the prox-
imal row in the longer one. In the following experiment the tip
was not removed until after the distal row of tentacles had appeared
(Fig. 20) The piece 4 was then removed and grafted in

Grafting of Tubularia 258

the reverse direction on the proximal piece (Fig. 24): This
brought the distal tentacles into a position which was slightly dif-
ferent from their normal one. ‘The space in front of them, 1. e.,
between them and the end was greater than before. In a large
number of experiments the pieces united, the tentacles completed
themselves, and the normal hydranth emerged. In about 10
per cent of the grafts, a most interesting result was observed. ‘The
original distal tentacle ridges remained as distinct bands, while
in front of them at the cut surface, a new row of distal tentacles and
hypostome developed, the hydranth finally emerged with the
stripes running from the base of the new distal tentacles to the
proximal ones (Fig. 25). No reproductive organs formed on
these pieces. The bands persisted until the hydranths dropped
off. This serves to demonstrate that the small distal piece A
when connected with the proximal piece is capable of developing
new structures after the tentacles have been laid down. It also
shows that the “red stuff” which is seen in the ridges, is not used
again when a new row of tentacles is laid down. Stevens (’02) and
Child (07c) have observed in the proximal piece, after separation
of the two ridges, that the “red stuff’? which was originally in
the proximal row forms a mass at the end of the stem, and when
the hydranth is completed the mass is ejected.

8 REMOVAL OF THE ENTIRE PRIMORDIUM

Driesch (’02) found when he removed the early anlage of the
hydranth by a cut just below the proximal row, that in a large
number of cases the ridges disappeared, and a new anlage devel-
oped, the latter being much reduced in length. Thus the length
of the “reparation area”? bore a definite relation to the length
of the piece. Child (’07a) has also shown that there is a reduc-
tion in length of the primordia in short pieces, but the reduction
in length is not proportional to the reduction in the length of the
piece.

I have made a series of experiments in which long pieces were
cut from the stems of different individuals, and after the tentacle
aniage appeared, the distal end of the piece was removed by a cut
below the proximal tentacles (Fig. 26, X). I hoped to find that at

354 Florence Peebles

a fixed distance below the primordium the stimulus of the cut
would result in the formation of a short aboral hydranth while the
oral ridges were fading out and the new short ones were appearing.
The results were as follows: When the space below exactly
equaled the length of the primordium (Fig. 26) the hydranth con-
tinued to develop without any sign of delay caused by the cut.
No hydranth developed on the aboral end for this region became
the stalk. When the space below the primordium was equal to
one-half the length of the primordium the result was the same.
The cut was then made close to the base of the proximal row of
tentacles. In about one-third of the pieces the original anlage
disappeared and a new one much shorter than the first appeared.
No aboral hydranths formed on these pieces.

un) Miz J hula i ni
ew) PP oe i

es ee

Fic.20 Fie.21 Fic.22 Fic.23 Fic. 24 Fic. 25 Fic. 26

Q THE INFLUENCE OF THE CONCENTRATION OF THE SEA-WATER

~ Loeb’s earlier work (’92).0n Tubularia brought to light the fact
that the concentration of the sea-water is a definite factor in the
regeneration of new hydranths. He found after testing various
strengths, that sea-water diluted to 66 per cent was the optimum
strength for growth. If the solution was weaker or stronger the
growth was retarded. Snyder (’05) has also tested various
strengths of sea-water, and has found that in Tubularia crocea
when the sea-water was diluted not only was greater growth
observed but a larger number of aboral hydranths developed.
My own experiments confirm those of Loeb and Snyder, but

Grafting of Tubularia 355

some of the results which I obtained indicate that the great increase
in size of the hydranths and the rapidity of their formation in
dilute sea-water is due to something more than the difference in
osmotic pressure.

The concentration of the sea-water in the Bay of Naples is
estimated at 3.8 per cent. If this water is diluted to 2.5 per cent,
growth is increased. Herbst (’04) found that artificial sea-water
of the same strength as that of the Bay of Naples was more favor-
able for growth of sea-urchin larvae than normal sea-water. I
followed Herbst’s formula and made a solution of artificial sea-
water. I found that growth in this solution was more rapid and
also that the hydranths formed in this solution were larger than
usual, and lived longer. When the artificial solution was diluted
with distilled water, the result was very different from that obtained
from diluted water from the Bay. ‘There was no increase in
growth or in rate of regeneration. ‘The results showed that the
solution was not as favorable for growth as normal sea-water.
I concluded from this that osmosis could not be the only factor
in determining the increase in growth. _ It is not altogether improb-
able that organic substances in the Bay of Naples exert a retard-
ing effect on growth. When these are excluded by preparing the
pure artificial sea-water their retarding influence is abolished, and
the growth which we consider so unusual is really no more than
the normal rate under optimum conditions. ‘This would explain
why diluting artificial sea-water does not produce the same result
as diluting that which comes directly from the Bay of Naples.

Child (’07c) has also made a study of the effect of diluting the
sea-water. He concludes that the diluted medium increases the
energy of the processes which involve hydranth formation. Since
I made my experiments with dilute sea-water Child’s work has
been published. As my results are practically the same as his
I shall omit a description of the experiments.

SUMMARY

1 When two individuals are grafted together changes at once
take place in the region of the graft. These changes may not be

356 Florence Peebles

visible externally, but they exert some influence on the rest of the
pieces, whether they result in the formation of new structures
or not.

2 When the aboral end of a piece of the stem of Tubularia is
stimulated through grafting to produce a hydranth before the oral
end, the change in the polarity is not lasting for when a second set
of hydranths develop, the piece assumes its original polarity.

3 When a short and a long piece are grafted together the
influence of the longer piece is shown only when the new hydranth
comes from the two pieces, a row of tentacles forming in each.

4 Short pieces grafted together in any direction usually develop
a hydranth on one of the free ends only, or first on one end and
later on the other. The result is the same whether the currents
in the two pieces are continuous or not.

5 Ifthe tip is removed from the end of a stem on which a new
hydranth has just begun to develop, before the ridges are visible,
the small piece is capable of forming a complete hydranth. If
the tip is removed after the ridges are laid down, the piece devel-
ops one row of tentacles. The proximal piece completes itself
distally by forming new tentacles on its tip, either before or after
emerging from the perisarc. If the tip is removed before the
primordia appear the proximal piece usually forms new primordia
without delay.

6 If, after the appearance of the primordia, the piece in which
the distal row of tentacles develops is reversed and grafted back
on the proximal row, the hydranth completes itself in the normal
manner. Sometimes the distal piece develops a new rowof distal
tentacles in front of the original red ridges which persist after the
hydranth emerges.

7 When the entire primordium is removed by a cut just below
the base of the proximal tentacles, the ridges frequently fade out
and a new primordium develops which is Fas shorter than the
original one. A cut 2 to 3 mm. below the primordium does
not affect its later development. Such pieces do not form aboral
hydranths.

8 Dhiluting normal sea-water in which pieces of Tubularia are
placed, increases the rate of growth and the percentage of new

Grafting of Tubularia 357

hydranths formed. Artificial sea-water has the same effect, but
when the artificial sea-water is diluted these favorable results
do not follow.

Bryn Mawr, Pa.
December 27, 1907

BIBLIOGRAPHY

Cuttp, C. M., ’07a—An Analysis of Form-Regulation in Tubularia. II. Differ-
ences in Proportion in the Primordia. Archiv f. Entw.-Mech.,
23, 1907.

o7b—An Analysis of Form-Regulation in Tubularia. V. Regulation
in Short Pieces. Archiv. f. Entw.-Mech., 24, 1907.

’o7e—An Analysis of Form-Regulation in Tubularia. VI. The Signif-
cance of Certain Modifications of Regulation: Polarity and
Form-Regulation in General. Archiv f. Entw-Mech., 24, 1907.

Driescu, H. ’97—Studien tber das Regulationsverméogen der Organismen. I.
Von den regulativen Wachthums und Differenzirungsfahig-
keiten der Tubularia. Archiv fiir Entwickelungsmechanik der
Organismen. 5. 1897.

’o2—Studien tiber das Regulationsvermogen der Organismen. 7
Zwei neue Regulationen bei Tubularia. Archiv f. Entw.-Mech.,
14, 1902.

Hersst, C., ’04—Ueber die zur Entwickelung der Seeigellarven nothwendigen
anorganischen Stoffe, ihre Rolle und ihre Vertretbarkeit. 3
Theil. Die Rolle der nothwendigen anorganischen Stoffe.
Archiv f. Entw.-Mech., 17, 1904.

Logs, J., ’92—Untersuchungen zur physiologischen Morphologie der Thiere.
11 Organbildung und Wachstum. Wurzburg, 1892.

’o4—Concerning Dynamic Conditions which Contribute toward the
Determination of the Morphological Polarity of Organisms.
University of California Publications, vol. 1, nos. 16 and 17,
1.904.

Morean, T. H., ’03—Some Factors in the Regeneration of Tubularia. Archiv
f. Entw.-Mech., 16, 1903.

Morean, T. H., and Stevens, N. M., ’04—Experiments on Polarityin Tubularia.
Journal of Experimental Zodlogy, vol. 1, 1904.

PEEBLES, ‘0O—Experiments in Regeneration and in Grafting of Hydrozoa. Archiv
f. Entw.-Mech., 23, 1900.

°o2—Further Experiments in Regeneration and Grafting of Hydroids.
Archiv f. Entw.-Mech., 14, 1902.

358 Florence Peebles

Snyper, C. D., ’05—The Effects of Distilled Water on Heteromorphosis in a Tubu-
larian Hydroid, Tubularia crocea. Archiv f. Entw.-Mech.,,
19, 1902.

Stevens, N. M., ’o2—Regeneration in Tubularia mesenbryanthemum. 11
Archiv f. Entw.-Mech 15, 1902.

A’ “sSLUDY

OF THE GERM CELLS OF CERTAIN

DIPTERA, WITH REFERENCE TO THE HETERO-
CHROMOSOMES AND THE PHENOMENA OF

SYNAPSIS

BY
N. M. STEVENS

With Four Pirates

INTRODUCTION

In connection with previous work on the spermatogenesis of
the Coleoptera (’05, ’06), the germ cells of the common fruit-fly,
Drosophila ampelophila, were examined in the autumn of 1906.
The difficulties encountered in handling this material led to the
study of the spermatogenesis of several other flies. The results
will be presented in accordance with the following scheme:

Calyptrate Muscidz.

Muscine.

t Musca domestica.
2 Calliphora vomitoria.
3 Lucilia cesar.

Sarcophagine. ,

4 Sarcophaga sarraciniz.

Anthomyiine.

5 Phorbia brassica.

Acalyptrate muscid.

6 Scatophaga pallida.
7 ‘Tetanocera sparsa.

8 Drosophila ampelophila.

Syrphide.

9g Eristalis tenax.

METHODS

With this material it was found that the best results could be
obtained from fresh tissue mounted in Schneider’s aceto-carmine.

Tue Journat or ExreriMeNTAL ZoOLoGy, VoL. v, No. 3

360 N. M. Stevens

The testes (or ovaries) of adult flies were dissected out in physio-
logical salt solution and immediately transferred to a drop of aceto-
carmine on a slide. The cover-glass was pressed down with a
needle to break the capsule of the testis and spread the cells. All
excess of stain was removed with filter paper, and after ten or
fifteen minutes, the preparation was sealed with vaseline. Such
preparations may be studied to the best advantage after twenty-
four hours, as the chromatin gradually acquires a deeper tint.
They remain in good condition for several days, but are, of course,
not permanent. The method has several advantages besides that
of enabling one to examine a large amount of material in a limited
time. The aceto-carmine fixes and stains instantly without the
shrinkage incident to the usual treatment with fixing fluids, alco-
hols, xylol and parafhn, necessary in order to obtain sections.
Then, one is able to study the whole cell with all of the chromo-
somes present and uncut, which is an obvious advantage for work
of this kind. The chromatin stains much more deeply than any
other cell element, but the achromatic structures are not always
well brought out, and they have been omitted from most of the
figures, as this investigation is concerned primarily with the hetero-
chromosomes and the method of synapsis. In favorable prepara-
tions of this kind, with good light, it is possible to get as accurate
camera drawings as from sections stained with iron hematoxylin.

RESULTS OF INVESTIGATION

I Musca domestica

In many respects the spermatogenesis of this fly resembles that
of Tenebrio molitor (Stevens ’05), Odontata dorsalis (Stevens
’06) and the other Coleoptera which have an unequal pair of
heterochromosomes. ‘There are, however, no synapsis, synize-
sis Or spireme stages in the spermatocytes, nor are tetrads ever
formed.

In the prophase of spermatogonial mitoses one finds five pairs
of long slender chromosomes, the members of each pair either
lying parallel to each other or twisted together (Fig. 1). The
members of the additional unequal pair are usually separate

The Germ Cells of Diptera 361

(Fig. 1, h, and h,). Apparently a side-to-side pairing or conju-
gation of homologous chromosomes, with the possible exception
of the unequal pair, occurs preliminary to each spermatogonial
mitosis. [he twelve chromosomes separate, and each divides
longitudinally in metakinesis. Whether they pair again in the
telophase or not until the prophase of another cell-division is not
evident.

The heterochromosomes remain condensed and are found side
by side during the whole growth stage, while the other chromo-
somes pass into a more or less diffuse condition (Fig. 2). In the
prophase of the first spermatocyte mitosis there are five thick
V-shaped chromosomes and a pear-shaped mass of chromatin
which in metakinesis proves to be the unequal pair of hetero-
chromosomes (Fig. 3). The V-shaped chromosomes all divide
longitudinally and the larger and smaller heterochromosomes
separate as seen in Fig. 4. In the interval between the first and
second divisions a nuclear membrane forms, but the chromosomes
do not change greatly. Figs. 5 and 6 show the two kinds of
daughter nuclei, one containing the larger, the other the smaller
heterochromosome. In the second spermatocyte mitosis the V-
shaped chromosomes again divide longitudinally and the hetero-
chromosomes divide as shown in Figs. 7 and 8, so that in all stages
they are clearly distinguishable from the ordinary chromosomes.
The resulting spermatozoa fall into two equal classes, dimorphic
as to the heterochromosomes, as in similar cases among the Hem-
iptera and Coleoptera. In most of the flies studied there was no
difficulty in finding odgonia in which the number and relative
size of the chromosomes could be determined. Only one such
was found in Musca, that shown in Fig. 9. Here a part of the
chromosomes are still paired; others have separated, but the
members of each pair of ordinary chromosomes are not far apart;
while the two equal heterochromosomes are on opposite sides of
the group (h). Here again we have what may be regarded as a
partial synapsis of homologous chromosomes. The relation of
the heterochromosomes in the two sexes is the same as in many of
the Coleoptera (Stevens ’05 and ’06) and the Hemiptera heterop-
tera (Wilson ’o5 and ’o6), an unequal pair (large and small) in

362 N. M. Stevens

the male and an equal pair of large heterochromosomes in the
female. An egg which is fertilized by a spermatozo6n containing
the smaller heterochromosome produces a male, while one which
unites with a spermatozoon containing the larger heterochromo-
some produces a female.

Although there is no distinct synapsis stage visible in the devel-
opment of the spermatocytes of Musca domestica, the method of
synapsis is without doubt indicated by the side-to-side pairing of
chromosomes of equal length in the prophases of both spermato-
gonial and odgonial mitoses. The final synapsis is a closer union
of the homologous chromosomes, and the first spermatocyte
division separates the members of each pair instead of dividing
each chromosome as in the spermatogonia.

2 Calli phora vomuitoria

The chromosomes in this species are similar to those in Musca
domestica. Both members of the unequal pair of heterochromo-
somes are smaller, as may be seen in a spermatogonial metaphase
(Fig. 10). Pairing of homologous chromosomes is also evident
here. In the growth stage (Fig. 11) the heterochromosomes are
associated with a plasmosome as in many species of Coleoptera.
Two views of the metaphase of the first spermatocyte mitosis are
shown in Figs. 12 and 13, and an anaphase in Fig. 14. Two
metaphases and an anaphase of the second division appear in
Figs. 15, 16 and 17. The equal pair of heterochromosomes in
the female is clearly shown in two odgonial metaphases (Figs.
18 and 19). In this case we have further evidence of the side-to-
side pairing of homologous chromosomes in the spermatogonia
and oogonia.

3 Lucilia Cesar

Only a few specimens of this species were captured and the
series of stages is incomplete. No spermatogonial or odgonial
metaphases were found. In the growth stage a pair of m-chro-
mosomes is present with an enormous heterochromosome bivalent

The Germ Cells of Diptera 363

(Fig. 20). The metaphase of the first spermatocyte division is
shown in Figs. 21 and 22, and prophases of the two kinds of second
spermatocytes in Figs. 23 and 24. The spermatozoa would evi-
dently be dimorphic as in the other species.

4 Sarcophaga sarracinie

The three species of Diptera whose spermatogenesis has already
been described belong to the sub-family Muscinz, while Sarcoph-
aga is a member of the sub-family Sarcophagine. The number
of chromosomes in Sarcophaga is the same as in the other species,
I2 somatic and 6 reduced, and the heterochromosomes closely
resemble those in Calliphora. The spermatogonial plate (Fig.
25) shows the 12 chromosomes paired, but separated ready for
metakinesis, and one chromosome shows the division line. In
the growth stage (Fig. 26) the pair of heterochromosomes comes
out clearly in the midst of diffuse and irregular masses of faintly
stained chromatin. In these flies the ordinary chromosomes
become much branched or diffusely granular in the growth stage
but do not unite to form a spireme of even width as in so many
forms. Whether or not they unite end-to-end at any stage before
or after synapsis I cannot say. A prophase and an equatorial
plate of the first spermatocyte mitosis may be seen in Figs. 27 and
28, and the metaphase and anaphase in Figs. 29, 30 and 31. The
polar views of the metaphase of the second mitosis (Figs. 32 and
33) of course show dimorphism as to the heterochromosomes
(Ay, h,). Equal division of all of the chromosomes follows as in
the three preceding species. Figs. 34 and 35 were drawn from
adjacent odgonia in metaphase to show the close longitudinal
pairing of the chromosomes and their later separation before
metakinesis. The equal heterochromosomes are usually found
together in the middle of the plate and each one is evidently equiv-
alent in size to the larger heterochromosome of the spermato-
gonia and spermatocytes (Figs. 25 to 33). Fig. 36 is from an
ovarian follicle cell. The four figures, 25, 34, 35 and 36 show the
pairing of homologous chromosomes in spermatogonia, O6gonia
and somatic cells.

304 N. M. Stevens
5 Phorbia brassica

Only one male of Phorbia was obtained and only four stages
drawn; but these indicate precisely the same conditions as in the
other species examined. Phorbia belongs to the sub-family
Anthomyine. Fig. 37, a growth stage; 38, a prophase; 39, a
metaphase; and 40, an anaphase, show clearly the presence of an
unequal pair of heterochromosomes resembling those of Musca
domestica.

6 Scatophaga pallida
7 Tetanocera sparsa

The chromosomes of Scatophaga and Tetanocera resemble each
other so closely in number, form and behavior that they will be
considered together. Fig. 41 is a spermatogonial prophase of
Scatophaga; and Figs. 42 and 43, spermatogonial prophase and
metaphase of Tetanocera. All show equally paired ordinary or
V-shaped chromosomes and unequally paired heterochromosomes.
Figs. 44 and 45 are prophase and metaphase of the first spermato-
cyte of Scatophaga, Figs. 46 and 47 the corresponding stages for
Tetanocera. In both species it will be seen that there is a close
resemblance between the paired condition of the chromosomes in
the prophases of a spermatogonial division and of a first spermato-
cyte mitosis. In general the chromosomes were larger in the
spermatogonia (Figs. 41, 42, 43) than in the spermatocytes (Figs.
44, 45, 46, 47), but frequently prophases of spermatocyte mitoses
could be certainly identifed as such only by the metaphases in
the same cyst and the growth stages in the neighboring cysts.
The only actual observable difference between the synaptic con-
dition in the spermatocytes and the spermatogonia is the behavior
of the pairs in the following mitosis: in the spermatogonia the
members of the pairs separate in metaphase (Fig. 43), and each
divides in metakinesis; while in the spermatocytes the members of
each pair remain closely associated in metaphase (Figs. 45 and 47)
and separate in metakineses (Fig. 48), but do not divide until the

The Germ Cells of Diptera 365

second spermatocyte mitosis, though they frequently show the
preparatory split in the anaphase (Fig. 49). We have here an
unusually clear demonstration of the essential facts of synapsis
and reduction, together with the rather unusual phenomenon of
conjugation of homologous chromosomes in cells outside the
sphere of maturation. Prophases of the second spermatocyte
mitosis in Scatophaga appear in Figs. 50 and 51, and metaphases
in Tetanocera in Figs. 52 and 53. An odgonial prophase and
anaphase are given in Figs. 54 and 55, and a late prophase for
Tetanocera in Fig. 56.

These two species as well as the one following belong to the
Acalyptrate Muscide.

8 Drosophila ampelophila

Drosophila has been placed at the end of the list of Muscidz
because of the peculiarities which occur in the behavior of its
chromosomes and the difficulties which have been encountered in
their interpretation. While in Sarcophaga all the stages neces-
sary for a description of the behavior of the heterochromosomes
of both sexes were found in the course of a few hours’ work on
perhaps ten or twelve preparations, satisfactory results in the case
of Drosophila have been obtained only after a prolonged study
extending over more than a year and involving the dissection and
examination of some two thousand individuals. Sectioning the
material has never given satisfactory results. Hermann’s platino-
osmic solution and Worcester’s formal-sublimate gave the best
fixation, but the division stages are so scattering that permanent
preparations, even if good fixation were secured, seemed less prac-
tical than the aceto-carmine method, which is much quicker and
gives clearer pictures of the mitotic phenomena when they are
present.

Spermatogonial mitoses are not abundant, and cells in which
perfectly clear equatorial plates can be studied are exceedingly
rare. [he chromosomes in prophase are paired and twisted
together in such a manner that it has been impossible to make an
intelligible drawing of them in an early prophase. In Fig. 57, a

366 N.M. Stevens

late prophase, two small spherical chromosomes and four larger
elongated ones are distinctly paired while the members of the
unequal pair (/,, h,) are separated. For a long time it was
impossible to be sure that an unequal pair was present, as fore-
shortening in the case of one chromosome (/,) was possible, but
recently a comparatively large number of good spermatogonial
plates has been secured in which the inequality in length of one
pair is clearly demonstrated. No case has been found in which
the members of this pair appeared to be equal. Figs. 58, 59 and
60 show exceptionally clear cases, and Fig. 61 shows a peculiar
folding of the chromosome /,, whose significance may be apparent
as we proceed to consider the unequal heterochromosome bivalent
of the first spermatocyte.

In Drosophila the heterochromosomes cannot be demonstrated
in the growth stages of the first spermatocyte. In some sections
from Hermann material stained with thionin the plasmosome
(p) and some of the chromosomes appeared as in Fig. 62 in cysts
adjacent to the spermatogonial cysts. In later growth stages
nothing definite, except the immense plasmosome, can be made
out in regard to the contents of the nucleus. The earliest pro-
phase of division is the appearance of the chromatin massed
together, usually on one side of the nucleus, while the plasmo-
some may be in the middle or on one side of the nucleus (Fig. 63).
In aceto-carmine preparations the chromosomes first appear in
early prophase, scattered through the nucleus, faintly stained and
irregular in outline (Fig. 64). The plasmosome may be broken
up at this time or it may appear intact in the spindle. Figs. 65 and
66 are later prophases in which the chromosomes are completely
condensed. The unequal heterochromosomes are h, and hy.
Fig. 67 shows the three equal bivalents, and the unequal hetero-
chromosome pair in its simplest form, in the metaphase of the
first spermatocyte mitosis. Fig. 68 shows slight modifications of
this form from other cells of the same cyst. The most common
form of this pair is seen in Figs. 69 and 70, where there are two
equal V-shaped elements and a third portion (x) which in many
cases looks like a separate element, and for a time the group was
thought to be trivalent; 1. e., made up of two equal V-shaped

The Germ Cells of Diptera mg07,

chromosomes and a smaller odd chromosome. ‘This belief was
strengthened by the appearance of many metaphases and ana-
phases (Figs. 70, 71, 72) where the third portion of the figure (x)
seemed to be on the point of separating from the V-shaped element
next to it. This opinion was not confirmed however by the
composition of the spermatogonial or oégonial equatorial plates,
nor was it possible to demonstrate with certainty a separate ele-
ment corresponding to x in the polar plates of the first spermato-
cyte mitosis or in the second spermatocyte. Fig. 72 1s one of
several cases where the portion x seemed to be separated from
the two other elements of the group, but the separation'must have
been only apparent, for one much oftener finds an anaphase like
Fig. 73 where the separation of the heterochromosome group into
two unequal parts is certain (iu, h,). Sometimes the anaphase is
like Fig. 74, where more or less spherical masses replace the usual
V’s of the heterochromosome group. Often all of the chromo-
somes except the smallest pair show in the metaphase that they
are elongated and V-shaped (Fig. 75), and in late anaphases
(Fig. 76) the elements of the two largest bivalents are usually
divided and the daughter chromosomes separated, often crossed.
Both here and in the second spermatocytes it is often difficult or
impossible to distinguish the heterochromosomes from the others.
In the telophase the chromatin forms a dense mass which loses
none of its staining quality and is soon resolved into the already
divided chromosomes of the second spermatocytes (Figs. 77, 78,
79). A greater or less degree of elongation together with twisting
and fore-shortening makes it impossible to measure or even esti-
mate with any accuracy the relative length of the chromosomes,
so as to distinguish the two classes of second spermatocytes as
to size of heterochromosomes. Figs. 78 and 79 are two equa-
torial plates from the same cyst where the corresponding chromo-
somes are probably a—a, b—b, and h,—h,. All of the chromo-
somes divide in this mitosis.

The odgonial metaphases are perfectly clear, and four equal
pairs of chromosomes are always present (Figs. 80, 81, 82). In
the metaphase they are usually grouped in pairs, and in the pro-
phase they are closely approximated and twisted. In fact this

368 N. M. Stevens

prereductional pairing of homologous chromosomes was first
noticed in the odgonia and ovarian follicle cells of Drosophila.
An attempt was made to ascertain whether such a pairing occurs
in embryonic cells. Very little evidence was obtained. In the
prophase of one mitosis paired chromosomes were found (Fig.
83). Fig. 84 is the equatorial plate of a segmentation stage. In
both cases the pairs appeared to be equal.

Q Eristalis tenax

A considerable number of these flies were captured on some
late blooming mustard plants in October. The material was in
exceptionally favorable condition, and a complete series of draw-
ings was obtained. ‘The outer wall, or capsule, of the testis was
thinner and more permeable to fixing fluids than in most of the
other species studied and it was therefore possible to work with
both sections and aceto-carmine preparations. ‘This fly belongs
to the family Syrphide, but the chromosomes in most respects
resemble those of the Muscide. The heterochromosome bivalent
is different in form from that of any of the Muscidz described
above; it however consists of a larger and a smaller component
united in a somewhat different way from the corresponding ele-
ments in Drosophila.

Among the spermatocytes, several follicle cells were found in
mitosis; the chromosomes of one such is shown in Fig. 85. The
spermatogonial chromosomes are paired in prophase but sepa-
rate and form a flat plate in the metaphase as seen in Fig. 86,
where the two heterochromosomes (/,, h,) are conspicuously
unequal in size. In this form there is a distinct synizesis stage,
as shown in Fig. 87, from a section of material fixed with Gilson’s
mercuro-nitric fluid and stained with thionin. ‘The cysts in which
this stage occurs border upon the spermatogonial region of the
testis. The outline of the chromosomes is visible and in the
next stage the chromosomes are distinctly bivalents. Later they
become more diffuse, but do not appear to form an even spireme
at any stage. Fig. 89 is a growth stage, showing the heterochro-
mosome group (/), a pair of m-chromosomes and the other chro-

The Germ Cells of Diptera 369

mosomes in a loosely branched condition. Fig. go ‘s an early
prophase in which the heterochromosome pair is very compact and
deeply stained, while the other chromosomes are granular and
denser in some parts than in others. A later prophase (Fig. 91),
from a section, shows the heterochromosome pair assuming the
cross-shape which we find in the later metaphase. Fig. 92 is a
polar view of the equatorial plate of the first spermatocyte; and
Figs. 93 and 94, side views of the spindle to show the cross-shaped
heterochromosome bivalent in two positions. Here the crosg
(Fig. 94), instead of having opposite arms equal, as in cross-
shaped tetrads composed of equal elements, has one of the ver-
tical arms longer. It is evident from Figs. 93 and g5 that the
longer arm is the smaller heterochromosome, while the remainder
of the cross is the larger member of the pair. The larger ele-
ment is folded in the same manner as in Drosophila (Figs. 66 and
67) but the smaller element is attached by one end instead of by the
middle as in Drosophila. The second spermatocyte mitosis pro-
ceeds as in the other forms and presents nothing of especial interest.
Dimorphism of the spermatozoa is foreshadowed by the first sper-
matocyte anaphases (Figs. g6 and g7). In the female the clearest
figures were obtained from ovarian follicle cells (Figs. g8 and 99).
The pairs are equal and comparison with the spermatogonial
chromosome group (Fig. 86) indicates that the equal heterochro-
mosome pair 1s one of the two longest.

The general results for the nine species of flies are the same; 1. e.,
an unequal pair of heterochromosomes in the male leading to
dimorphism of the spermatozoa, and a corresponding equal pair
in the female, each equivalent to the larger heterochromosome of
the male: also a prereductional pairing of homologous chromo-
somes in the prophase of mitosis in spermatogonia, o6gonia, and
ovarian follicle cells.

DISCUSSION

rt Sex Determination

So far as I know there is no published work on the heterochro-
mosomes of the Diptera. The literature on the heterochromo-

37° N.M. Stevens

somes in other orders of insects has recently been so fully dis-
cussed in a paper by A. M. Boring (’07) that it seems hardly nec-
essary to go into the subject exhaustively here. “The dimorphism
of the spermatozoa resulting from the maturation of the male
germ cells of the nine species of Diptera considered in this paper
is of the same character as that described by the author for 36
species of Coleoptera (see note, p. 49, Stevens ’06), and by Wilson
(05 and ’o6) for several species of Hemiptera heteroptera. The
dimorphism is brought about by the presence in the spermatogonia
and spermatocytes of an unequal pair of heterochromosomes,
while in large numbers of other insects such dimorphism is due to
the presence of an odd chromosome in the male germ cells. These
flies have proved to be exceptionally favorable material for demon-
strating the occurrence in the female germ cells and somatic cells
of a pair of chromosomes, each equivalent to the larger hetero-
chromosome of the male.

Here, as in similar cases previously described, it is perfectly clear
that an egg fertilized by a spermatozo6n containing the smaller
heterochromosome produces a male, while one fertilized by a
spermatozoon containing the larger heterochromosome develops
into a female. The material does not, however, throw any fur-
ther light on the question whether the dimorphic spermatozoa
are themselves in some way instrumental in determining sex in
these insects; or whether sex is a character borne by the hetero-
chromosomes and segregated in the maturation of the germ cells
of each sex. If the latter supposition is true, sex is probably
determined by the dominant heterochromosome of the egg, and
fertilization is selective as has been shown in previous papers
(Wilson ’o5, ’06; Stevens ’06, p. 54; Nowlin ’06; Boring ’o7).

The only hope of determining whether sex is a Mendelian char-
acter seems at present to lie in breeding experiments with forms
that may be shown by cytological study to be favorable. It is
probable that in some cases at least, other characters may be so
correlated with sex that their behavior in heredity may throw
light on the sex question.

As to the proportion of sexes in these flies, a few figures may be
given for Drosophila ampelophila. In the autumn and winter

The Germ Cells of Diptera 371

of 1906-07, Drosophila was bred in the laboratory on two kinds of
food, grapes and bananas. As the flies were dissected for the
cytological work, a record was kept of the numbers of each sex;
1551 were so recorded. Of these 759, or 48.94 per cent were
males; 792, or 51.06 per cent females. ‘The records of the grape-
fed and the banana-fed flies were kept separately. The total
number of grape-fed flies dissected between November 1 and
March 19 was 787, 404 or 51.33 per cent being males, and 383 or
48.67 per cent females. The banana-fed flies between October
30 and December 3 numbered 764, 355 or 46.47 per cent males,
and 409 or 53.53 per cent females. In the total number there
were 2.02 per cent more females than males, in the grape-fed
2.66 per cent more males than females, and in the banana-fed
7.06 per cent more females than males. These differences are
probably not significant, but if sex is a Mendelian character, the
numbers for the two sexes should of course be equal unless food
produces some discriminating effect on the development of either
individuals or eggs of the different sexes. It has always been a
noticeable fact that the banana-fed flies were larger and more
robust than those fed on grapes; this however applies to both
sexes. In mass cultures it is not possible to tell whether failure
of many of one sex or the other to reach the adult stage in differ-
ent cultures might account for the discrepancies in numbers
observed with the two kinds of food.

Castle and his co-workers (’06 p. 772) found the sexes about
equal in three families of the sixth inbred generation of a grape-
fed series, and the remarks which follow the table indicate that
they regard the normal proportion as near equality.

Monkhaus’ results on sex in Drosophila seem not yet to be in
print, except for a brief report in the Year Book of the Carnegie
Institution.

An attempt was made to ascertain the normal proportion of the
sexes for the adults of Musca domestica. When caught by hand
58.33 per cent were females, but when a wire trap baited with
sugar or molasses was used, only 46.53 per cent were females.
The results need no comment.

Cuénot states that the normal proportion of males to females

372 N.M. Stevens

in Lucilia caesar, Calliphora vomitoria and Sarcophaga carnaria
is approximately equal, and his experiments show that neither
amount nor kind of food given to the larvae has any marked effect
on the proportion of the sexes in the first or second generation, but
here as elsewhere in such experiments the number of eggs that
did not hatch is not noted, and this may be the critical point.
It is evident that more experiments are needed in which the fate
of all of the eggs of isolated pairs of flies is determined.

2 Synapsis

In the spermatogenesis of most insects synapsis involves an
end-to-end union of homologous chromosomes, and tetrads of
various forms are commonly found in the prophase of the first
spermatocytes. In these flies no tetrads have been observed and
as a rule nothing comparable to the synizesis, bouquet or spireme
stages of other forms is apparent. In these respects the germ
cells of the Diptera resemble the oogonia of sagitta (Stevens ’03
and ’o5) and the male and female germ cells of the aphids (Stevens
’o5 and ’o6). In the oogonia of Sagitta the chromosomes pair
side-to-side in an early stage, while in the spermatogonia of the
aphids the pairing occurs as a prophase of the first spermatocyte
mitosis. ‘The indications are that in the flies the chromosomes
are already paired side-to-side at the beginning of the growth
stage (Figs. 87 and 88), but the pairs do not appear to unite end-
to-end to form a spireme. In some cases the members of the pairs
are perfectly fused in the prophase of the first spermatocyte (Figs.
3 and 27); 1n others the bivalents are clearly such in both prophase
and metaphase (Figs. 44 to 46). The first spermatocyte division
is without doubt reductional for both ordinary chromosomes and
heterochromosomes.

Perhaps the most interesting point in the whole study is the
pairing of the chromosomes in cells somewhat removed from the
sphere of the reduction process. This was first noticed in the
oogonia of Drosophila, and was also found to occur in the ovarian
follicle cells, the spermatogonia and some embryonic cells. This
is not an occasional phenomenon, but one which belongs to every

The Germ Cells of Diptera 292

odgonial and spermatogonial mitosis. In many cases the pro-
phases of spermatogonia and first spermatocytes resemble each
other very closely, the members of each pair being twisted
together in both. In the spermatocyte we get complete synapsis
and reduction; in the spermatogonium only a foreshadowing of
reduction, and abundant proof that synapsis is here a side-to-side
pairing of homologous chromosomes, and the first spermatocyte
division a separation of univalent chromosomes, and not a
longitudinal or quantitative division of two chromosomes united
end-to-end. ‘The relation of the heterochromosomes to each other
in synapsis varies greatly with differences in form and size.

One is tempted to suggest thatif homologous maternal and pater-
nal chromosomes in the same cell ever exert any influence on each
other, such that it is manifest in the heredity of the offspring,
there is more opportunity for such influence in these flies than in
cases where pairing of homologous chromosomes occurs but once
in a generation. Possibly experiments in cross-breeding of flies
may bring out some interesting facts in heredity.

Nore. A preliminary statement in regard to the chromosomes of Drosophila
was made at the International Congress of Zodlogists in Boston, August 21, 1907.
The question as to whether an odd chromosome or an unequal pair of heterochro-
mosomes was present in the male cells was then unsettled.

LITERATURE CITED

Borine, A. M., ’07—A Study of the Spermatogenesis of Twenty-two Species of
the Membracidw, Jassida, Cercopida and Fulgoride. Jour.
Exp. Zodl., vol. iv.

Castie, W. E., and others, ’06—The Effects of Inbreeding, Crossbreeding, and
Selection upon the Fertility and Variability of Drosophila.
Mus. Comp. Zoél., Harvard, no. 177.

CuEnor, L., ’99—Sur la détermination du sexe chez les animaux. Bull. Sci. de
la France et Belg., vol. xxxii.

Nowuin, W. N., ’06—A Study of the Spermatogenesis of Coptocycla aurichalcea
and Coptocycla guttata, with especial reference to the Problem

of Sex Determination. Jour. Exp. Zodl., vol. iii.

374. N. M. Stevens

Stevens, N. M., ’03—On the Ovogenesis and Spermatogenesis of Sagitta bipunc-
tata. Zool. Jahrb., vol. xviii.

’o5—Further Studies on the Ovogenesis of Sagitta. Zool. Jahrb., vol. xxi.

‘o5—A Study of the Germ Cells of Aphis rose and Aphis cenothere.
Jour. Exp. Zol. vol. 11.

’o5—Studies in Spermatogenesis with especial reference to the “Accessory
Chromosome.” Carnegie Inst., Wash., Pub. 36. — ;

’o6—Studies on the Germ Cells of Aphids. Carnegie Inst., Wash., Pub.
5.

‘o6—Studies in Spermatogenesis. I]. A Comparative Study of the
Heterochromosomes in Certain Species of Coleoptera, Hemiptera
and Lepidoptera, with especial reference to Sex Determination.
Carnegie Inst., Wash., Pub. 36, no. 2.

Witson, E. B., ’0o5—Chromosomes in Relation to the Determination of sex in
Insects. Science, n. s., vol. xx.

’o5s—Studieson Chromosomes. I. ‘The Behavior ofthe Idiochromosomes
in the Hemiptera. Jour. Exp. Zodl., vol. 1.

‘o5—Studies on Chromosomes. II. The paired Microchrosomes,
Idiochromosomes, and Heterotropic Chromosomes in the Hem-
iptera. Jour. Exp. Zodl., vol. il.

*o6—Studies on Chromosomes. II. Sexual Differences of the Chromo-
some Groups in Hemiptera, with some Considerations on
Determination and Inheritance of Sex. Jour. Exp. Zodl., vol. i.

Pirate IL

Sarcophaga sarracinia

Fig. 25 Spermatogonium, metaphase.

Fig. 26 First spermatocyte, growth stage.

Fig. 27 First spermatocyte, prophase.

Fig. 28 First spermatocyte metaphase.

Fig. 29 First spermatocyte, metakinesis.

Figs. 30 and 31 First spermatocyte, anaphase.
Figs. 32 and 33 Second spermatocyte, metaphase.
Figs. 34 and 35 Odgonia, metaphase.

Fig. 36 Ovarian follicle cell, metaphase.

Phorbia brassica

Fig. 37 First spermatocyte, growth stage.
Fig. 38 First spermatocyte, prophase.
Fig. 39 First spermatocyte, metaphase.
Fig. 40 First spermatocyte, anaphase.

Scatophaga pallida and Tetanocera sparsa

Fig. 41 Scatophaga, spermatogonium, prophase.
Fig. 42 Tetanocera, spermatogonium, prophase.
Fig. 43 Tetanocera, spermatogonium, metaphase.
Fig. 44 Scatophaga, first spermatocyte, prophase.
Fig. 45 Scatophaga, first spermatocyte, metaphase.

Pirate II

Scatophaga and Tetanocera (continued)

Fig. 46 Tetanocera, first spermatocyte, prophase.

Fig. 47 Tetanocera, first spermatocyte, metaphase.

Fig. 48 Scatophaga, first spermatocyte, anaphase.

Fig. 49 Scatophaga, first spermatocyte, anaphase.

Figs. 50 and 51 Scatophaga, second spermatocyte, prophase.
Figs. 52 and 53 Tetanocera, second spermatocyte, metaphase.
Fig. 54 Scatophaga, odgonium, prophase.

Fig. 55 Scatophaga, odgonium, anaphase.

Fig. 56 Tetanocera, odgoniaum, prophase.

Drosophila ampelophila

Fig. 57 Spermatogonium, late prophase.

Figs. 58-61 Spermatogonia, metaphase.

Fig. 62 First spermatocyte, early growth stage.

Fig. 63 First spermatocyte, very early prophase.
Fig. 64 First spermatocyte, prophase.

Figs. 65 and 66 First spermatocyte, late prophase.
Fig. 67 First spermatocyte, metaphase.

Fig. 68 Heterochromosome pairs.

Figs. 69-71 First spermatocyte, metaphase.

Figs. 72 and 73 First spermatocyte, anaphase.

PLATE III

a®,.

ie ei a = A we.

Parte IV

Drosophila (continued)

Fig. 74 First spermatocyte, anaphase.

Fig.75 First spermatocyte, metaphase.

Fig. 76 First spermatocyte, anaphase.

Figs. 77-79 Second spermatocyte, metaphase.
Figs. 80-82 Odgonia, metaphase.

Fig. 83, Chromosomes from embryonic cell.
Fig. 84. Chromosomés from segmentation stage.

Eristalis tenax

Fig. 85 Chromosomes of follicle cell of the testis.

Fig. 86 Spermatogonium, metaphase.

Fig.87 First spermatocyte, synizesis stage.

Fig. 88 First spermatocyte, growth stage immediately following synizesis stage.
Fig. 89 First spermatocyte, later growth stage.

Fig. 90 First spermatocyte, early prophase.

Fig. 91 First spermatocyte, late prophase.

Figs. 92-95 First spermatocyte, metaphase.

Figs. 96 and 97 First spermatocyte, anaphase.

Figs. 98 and 99 Chromosomes of ovarian follicle cells, prophase and metaphase

THE GERM CELLS OF DIPTERA PLATE IV

M. Srevens

|
y V
74 e vt "a R. a Y 42
>) abe or rN
h. AA 76 80 81 82
a a
mm ae te Od
y gm &e gy
A oh
A pe eo L ea 5 et

From the Marine Biological Laboratory, Wood's Hole, and the Laboratory of Physiological Zodlogy,

University of Pennsylyania

MOMENTARY ELEVATION OF TEMPERATURE AS
A MEANS OF PRODUCING ARTIFICIAL PARTHE-
NOGENESIS IN STARFISH EGGS AND THE CON-
DITIONS OF ITS ACTION

RALPH S. LILLIE

I INTRODUCTION

Exposure of mature eggs of Asterias forbesii to the influence of
cold sea-water (about 4° to 7°) for somewhat prolonged periods
(1 to 7 hours) was first shown by Greeley,! at Wood’s Hole in 1901,
to be followed by cleavage and production of larvae on return
to normal temperatures. Greeley also experimented with tem-
peratures higher than the normal, exposing eggs (taken from the
same dishes as those used for experiments with cold) to tempera-
tures of 31° to 37° for similar periods of time (1 to 7 hours); but
the results of this treatment were purely negative, the eggs merely
absorbing water and undergoing a change which he described as
liquefaction. He concluded, somewhat sweepingly, that “seg-
mentation of the starfish egg cannot be produced by raising the
temperature of the sea-water.” He found later (summer of 1902)
that temperature was an important factor in the production of
parthenogenesis by hypertonic solutions,’ the time of exposure
decreasing (within a certain range of temperatures) as the tempera-
ture rose, a result confirmed by Lyon* at Naples in the fall of
1902 for species of Strongylocentrotus and Arbacia. But eleva-
tion of temperature alone, unaccompanied by other treatment,
remained ineffective; moreover, “at 30° it was found impossible
to produce artificial parthenogenesis in Asterias or Arbacia with
any of the solutions used.”’ In the earlier paper Greeley had

1 Greeley: American Journal of Physiology, vi, p. 296, 1902.

2 Greeley: Biological Bulletin, iv, p. 129, 1903.
3 Lyon: American Journal of Physiology, ix, p. 308, 1903.

Tue JourNnaL or ExPERIMENTAL ZOOLOGY, VOL. V, NO. 3

76 Ralph S. Lillie

treated with incredulity Delage’s account of successful experiments
with higher temperatures, ascribing the results to the effects of
agitation and not of simple elevation of temperature. In Greeley’s
own experiments “when great care was exercised in handling the
eggs not a single segmentation was produced.” ‘The criticism,
however, was Te funded for it was clear from Delage’s papers’
that his eggs were exposed to the high temperatures at a time—
namely, e BG, maturation—when agitation is quite ineffective
in producing parthenogenesis. It is not until the eggs have been
mature for some time that this result appears;? while warming, as
Delage expressly afirms, is most effective during early matura-
tion stages. In Greeley’s second paper he again cites Delage’s
experiments, but without comment. Evidently his intention was
to return to the subject.

Since the appearance of Delage’s papers in 1go1 there seems
to have been little further investigation of the influence of rise of
temperature in exciting celepme of unfertilized eggs. “The
theoretical possibility that development could thus be iaceed was
incidentally adverted to by Loeb® some years later: if the sperma-
tozoon acts by introducing positive catalysers into the egg, thus
accelerating the chemical processes on which the initiation of devel-
opment depends, a similar acceleration with similar consequences
ought to follow simple elevation of temperature. Loeb has also
more recently emphasized the importance of the temperature
factor in the production of parthenogenesis by the use of hyper-
tonic solutions.* But no further experimental contributions have
appeared toward the solution of the question whether—and under
just what conditions—elevation of temperature can in itself initiate
the development of unfertilized eggs. The a prior: probability
that such would be found to be the case must have seemed strong
when the high temperature-coefiicient of the acceleration of chem-
ical processes was considered: a five or sixfold acceleration of at
least certain of the reactions occurring in the egg-substance would

‘Delage: Comptes rendus, cxxxiii, p. 348, 1901; Archives de zoologie expérimentale et générale,
3me Sér., ix, p. 285, 1gor.
5 Mathews: American Journal of Physiology, vi, p. 142, 1902.

6 J. Loeb: University of California Publications, Physiology, vol. ii, p. 158, 1905.
7 J. Loeb: Biochemische Zeitschrift, vol. 1, p. 183, 1906.

Artificral Parthenogenests in Starfish Eggs Biel
follow warming to 35° or 40°; and a fundamental change in the
properties of the system and possibly a removal of the conditions
impeding spontaneous development might reasonably be expected
to result from such treatment.

The failure of investigators in this country to obtain partheno-
genesis by elevation of temperature appears the less accountable
since Delage’s descriptions are at least sufficiently definite to have
suggested a procedure quite different from the one which was
actually employed and proved ineffectual. “Thus Delage writes®
“La température peut, a elle seul, surtout appliquée brusquement
a un stade critique, dont il va étre question, déterminer la parthéno-
génése chez Asterias.”’ This critical stage is described as the
time (approximately) at which the nuclear membrane of the ger-
minal vesicle disintegrates allowing the nuclear contents to enter
the cytoplasm; this event determines the time at which “merogonic”’
fertilization becomes possible, and also artificial parthenogenesis
by heat: “at this moment the eggs of Asterias can be made to
develop parthenogenetically by simple immersion in water warmed
to 30° to 33°."" The lack of exactitude in this description con-
sists chiefly in the failure to assign any definite limit of time to the
action of the warm sea-water. As will be seen below, this is a
matter of importance, since too long and too brief exposures alike
fail to produce the desired effect and lead simply to abnormal
changes resulting in the early disintegration of the egg. It is
clear, however, that the eggs in Delage’s experiments were warmed
for only a short period; in fact, he recommends placing the eggs
in warm sea-water contained in small vessels (cuvettes) which
may be rapidly cooled in running water.!? In Greeley’s experi-
ments the eggs (1) were allowed to mature—a necessary condition
for the production of parthenogenesis by cold, action of acids,
agitation, or hypertonic solutions, but one which precludes the
possibility of development by simple warming (as will be seen
below); and (2) were exposed to the high temperatures for periods
of an hour or more; whereas exposure to 35° for 60 or 70 seconds

* Delage: Comptes rendus, cxxxiii, p. 348, 1901.
® Delage: Comptes rendus, /oc. cit., p- 348.

10 Delage: Archives de zoologie expérimentale et générale, Joc. cit., p. 309.

378 Ralph 8. Lillie

is sufficient, at the proper time during the maturation period,
to produce development. It is not surprising that the eggs failed
to develop under these conditions. The general outcome of
Greeley’s own work on the influence of temperature changes on
protoplasm appears to have led him to doubt the possibility of
producing parthenogenesis by elevation of temperature. He had
found that cold, by inducing loss of water, exercises on protoplasm
an action similar to that of a hypertonic solution, which was already
known to produce parthenogenesis; and it must have seemed to
him scarcely possible that warmth, which affects the protoplasm
in a precisely opposite manner from cold, could have the same influ-
ence on the developmental process. It is also evident that his
experiments on the action of high temperatures in inducing par-
thenogenesis were less complete than those made with cold;
evidently his studies of the influence of temperature-conditions on
development were cut short while they were yet unfinished.

II EXPERIMENTAL

My own experiments were begun in the summer of 1906, at a
time when | was unaware that Delage had succeeded in producing
development by this means. ‘The idea with which the study was
begun was that possibly a slight change in the aggregation-state of
certain of the protoplasmic colloids might be a determining condi-
tion of development, and that such a change might be induced by a
momentary heating of the eggs. Heat coagulation produced by
momentary heating followed by rapid cooling was, according to
Corin and Ansiaux, a reversible process." Such a slight and re-
versible coagulation might conceivably without injuring the egg so
change the state of the egg substance as to cause development to be
resumed. It soon became evident, however, that even transitory
exposure to temperatures of 45° to 50°, the lowest at which heat
coagulation could be expected, was rapidly injurious, inducing
breakdown of the eggs without any developmental changes. On
the other hand, brief exposure to temperatures of 35° to 38°—in

4 Corin and Ansiaux: Bulletin de l’académie royale de Belgique, xxi, p. 345, 1891. The results
of Corin and Ansiaux have since been rendered doubtful by Pauli: Beitrage zur chemischen Physiologie

und Pathologie, x, p. 53, 1907.

Artificial Parthenogenesis in Starfish Eggs 379

general the optimum for enzyme action—gave extremely promising
indications. ‘The remainder of the investigation was then devoted
to determining the influence of such temperatures acting for vari-
ous brief periods.

In the following experiments the eggs were exposed for brief
periods (varying from a fewsecondsto several minutes) to the action
of sea-water previously warmed to a definite temperature. ‘The
procedure employed is as follows: the eggs are transferred at
a known period after removal from the animal to a small beaker
in which a thermometer is placed; sea-water at a temperature
slightly above that selected for the particular experiment (e. g., 35°)
is then added rapidly to the small beaker in quantity sufficient to
bring the temperature to the desired point; this temperature is
maintained constant during the definite time-period of the experi-
ment by partly immersing the small beaker, whenever necessary,
in a larger vessel of water at somewhat higher than the experi-
mental temperature. After the lapse of the selected time-period
(e. g., 70 seconds) the contents of the small beaker are suddenly
transferred to a large volume of sea-water at normal temperature
contained in a finger-bowl. The temperature of the eggs is thus
suddenly reduced again to the normal. It may safely be
assumed, when one considers the small volume of each egg and
the correspondingly large surface for thermal interchange with the
medium, that during at least the greater part of the period of
immersion in the warm sea-water the eggs have themselves been
at the same temperature as the medium. ‘The agitation involved
in the two transfers is unavoidable with this procedure; but at the
stages with which I have worked—mostly early maturation—
mechanical shock is in itself ineffective in causing development.
Mere transfer from one dish to another produces no visible result.
The effects observed are therefore to be ascribed wholly, or at
least inchief part, to the change in the thermal conditions prevailing
in the egg-system.

Experiments with Arbacia Eggs

The results with sea-urchin eggs have been almost entirely
negative so far as concerns production of development by momen-

380 Ralph S. Lillte

tary elevation of temperature. In the earliest experiments, eggs
were exposed for a few seconds to temperatures supposedly high
enough to cause partial coagulation of a portion of the colloidal
constituents of the protoplasm. “Temperatures of 45°, 50°, 55°,

and 60° were allowed to act for periods ranging from 5 to 60 sec-
onds. No noteworthy changes followed such treatment; swelling
and disintegration resulted from exposure for even brief periods to
the higher temperatures. A few eggs showed membranes similar
to fertilization membranes after exposure to 45° for a few seconds,
and occasionally some cleavages were found. The great majority
of eggs so treated died without showing any developmental change.

Treatment that resulted favorably with Asterias eggs also gave
imperfect or negative results with Arbacia. Eggs were'exposed to
35°. 37-5°, and 40°, for periods ranging from five seconds to
two minutes. In the most favorable experiments a few eggs
showed membranes and irregular cleavages; but development
never proceeded beyond a stage of a few cells, and the great major-
ity of eggs always remained apparently unaffected. I have also
attempted to induce cleavage in unfertilized Arbacia eggs after the
artificial production of a fertilization membrane by the method
introduced by Loeb, viz: treatment for one to two minutes with
a mixture of 3 cc. 3, acetic acid and 50 cc. sea-water. Eggs so
treated become, as in the case of Strongylocentrotus investigated
so thoroughly by Loeb, far more susceptible to the develop-
ment-inducing action of hypertonic sea-water; but the results after
warming to 35° for periods of 20, 30, 40, 60 and go seconds, within
10 to 15 minutes after membrane-formation, were in no observable
respect different from those obtained with the same eggs after
simple treatment with acidulated sea-water without warming. A
certain proportion of such eggs always undergo cleavage, usually
irregular, but development rarely proceeds farther than an early
stage ofafewcells. —

A striking phenomenon, which I have frequently observed in
sea-urchin eggs treated in the above manner with acidulated sea-
water, seems entitled to special mention here, namely, the appear-
ance of active amoeboid movements of the egg-protoplasm, at times
surprisingly energetic in character. “he movement appears most

Artificial Parthenogenests in Starfish Eggs 381

active about three or four hours after treatment with the acid-
ulated sea-water. The following record will illustrate:

July 15, 1907, 12.37 p.m. Unfertilized sea-urchin eggs were placed in a mixture of 50 cc. sea-water

+ 3c. N. acetic acid; one portion (A) was transferred to normal sea-water after one minute, a second

(B) after 1m. 30s. At 4:30 p. m., lot A showed numerous irregularly shaped eggs in which
active amoeboid movement was in progress. In many eggs the movement was so energetic that the
actual contractions of the cell-surface and the protrusion of pseudopodia were plainly visible; many
even exhibited an active crawling or squirming movement,suggestive of sluggish muscular contractions.
In many eggs small portions of the surface protoplasm were constricted off—small beadlike protuber-
ances like polar bodies being especially numerous. Transitions between irregular ameeboid masses and
distinct though irregular cleavage stages were not uncommon; the latter also showed continual and
active changes of form. Lot B showed essentially similar conditions. The temperature of the water in

the dishes was 25°.

This observation seems interesting on account of the unusually
active nature of the amceboid movements. “The assumption of
irregular amoeboid forms by various eggs is familiar to most
experimentalists,” and is especially frequent in starfish eggs. But
active crawling movements of the above kind have, so far as | am
aware, not hitherto been described in these eggs. ‘The theoretical
interest of the phenomenon consists chiefly in the very clear indica-
tion which it affords that the form-changes in cleavage are of essen-
tially the same nature and due to the same conditions as are the
ordinary amceboid movements of cells; these last, as may be inferred
from the closeness with whichthey may be artificially simulated, are
almost certainly due—at least as regards their main features—to
local (possibly electrically conditioned) changes of surface tension.
The above transitional condition between amceboid movement and
cleavage supports strongly the view that the change of form in
normal cell-division is also due to surface-tension changes, which
differ from those causing amceboid movements only in the very
regular and symmetrical distribution of the areas of lowered sur-
face tension.

Experiments with Starfish Eggs
A Conditions of Formation of Fertilization-membrane

Exposure to temperatures of 45° and higher caused mature star-
fish eggs to become coarse and opaque within 20 minutes or less.

” Especially energetic amoeboid movements are seen in abnormally developing parthenogenetic eggs
of Chetopterus; cf. F. R. Lillie, Archiv f. Entwicklungsmechanik, xiv, p. 487, 1902.

82 Ralph S. Lille

No membrane was formed. In one series of experiments, eggs
in early maturation stages (at which time membranes are most
readily formed) formed in some instances membranes on exposure
to 45° for 15 seconds; exposure to the same temperature for 30
seconds was followed by breakdown without membrane-formation.
Temperatures of 45° and higher are thus rapidly destructive, as
in the case of sea-urchin eggs; but very brief exposures may produce
some of the effects (as membrane-formation) of more favorable
conditions.

Temperatures of 40° and lower were then tried. The earliest
visible effect of brief warming at such temperatures is the forma-
tion of the fertilization membrane. The production of this mem-
brane appears to be associated with the removal of certain hin-
drances to further development (p. 385), and accordingly it may
be regarded as the first visible sign of developmental changes
in the egg. The structure is produced with remarkable ease by
momentary exposure of eggs to the action of warm sea-water; yet
it is significant that temperatures above a certain maximum (ca,
45°), acting for more than a few seconds, fail to cause its produc-
tion. Apparently some ferment-action, rather than the direct
effect of the heat, is concerned. It also fails to be produced at 30°
unless possibly the exposure is very prolonged. I have made few
observations with temperatures lower than 35°. The temperature
relations of this phenomenon ought perhaps to be more thoroughly
investigated.

The following table summarizes the results of three series of
experiments covering a considerable range of temperatures. “They
illustrate very typically some of the conditions of membrane-pro-
duction in starfish eggs.

Artificial Parthenogenesis in Starfish Eggs

TABLE I
Series I. Fuly 30, 1906

383

| RESULT
Time of |
Temperature exposure |
degrees A Eggs warmed before separa~| B Same eggs warmed four hours
eae tion of first polar bodies after removal from animal
|
30 15 | No membranes formed
30 | No membranes formed |
35 15 | No membranes | No membranes formed
30 | Membranes in more than half A few membranes
40 15 All form membranes Most form membranes
3° | All form membranes Almost all form membranes
45 15 All form membranes Almost all form membranes
30 None form membranes; eggs soon No membranes; eggs soon dis-
disintegrate integrate
50 15 No membranes; early disintegra-
| tion
30 | No membranes; early disintegra-
tion
Series II. August 1, 1906
RESULT
Temperature = = =
degrees Expose A Eggs warmed during mat- B Warmed 24 hours after
uration process completion of maturation
33 I5s. No membranes | Practically no membranes
305. No membranes Practically no membranes
6os. Almost all form membranes A few membranes
2m. Almost all form membranes A few membranes
35 15s. A few imperfect membranes Considerable number membranes
305. Most form membranes Practically all form membranes
1and2m. | All form membranes All form membranes
37-5 5s. A few imperfect membranes A few membranes
15s. Practically all form membranes| Practically all form membranes
30S. All form membranes Practically all form membranes
Im. All form membranes Practically all form membranes
40 5s. Practically all form membranes
15 and 30s.
All form membranes

and 1m.

384 Ralph S. Lillie

TABLE I—Continued

Series III. August 6,1906. Eggs warmed during maturation process

Temperature

In general the above observations may be regarded as typical,
though I have found some variability in the readiness with which
eggs from different animals form membranes. But with star-
fish eggs in the early maturation period membrane-formation rarely
or never fails if eggs are exposed to temperatures between 33° and
40° for the periods indicated as optimal in the above table. The
result is remarkably constant, even if the subsequent cleavage and
development should prove abnormal or should altogether fail.
The facility with which the membrane is produced varies also in
eggs from the same animal at different intervals after removal;
in general the early maturation stages, before the first polar body
is separated, are most favorable; after the completion of matura-
tion, membrane-production is less regular and constant, and more
prolonged exposures to the high temperature are necessary. This
change is possibly to be correlated with the change in suscepti-
bility to parthenogenetic development under this form of treatment,
which also diminishes after maturation is completed, as [ shall
describe later.

The minimum time of exposure necessary for membrane-pro-
duction is shown by the above experiments to decrease rapidly with
rise of temperature until a certain limit is reached. At 33° expo-
sure must be prolonged to two minutes: at 34° the minimum lies
somewhere between 30 and 60 seconds; at 35° between 15 and 30

Artificial Parthenogenesis in Starfish Eggs 385

seconds; at 37.5° between 5 and 15 seconds, and at 40° momen-
tary exposure (5 seconds) produces membranes in practically all
eggs. [hese temperature-relations point to an underlying process
hae undergoes unusually rapid acceleration with rise of tempera-
ture, until a certain optimum is reached (apparently in the neigh-
borhood of 40°), after which heat acts unfavorably. Exposure
to 45° for 30 seconds fails, as seen above, to produce membranes
and acts destructively on the eggs, although briefer exposure (15
seconds) may be effective.

The actual separation of the membrane may be readily studied.
Within to to 15 minutes after return to normal sea-water it appears
as a wavy or crenated layer adhering closely to the egg-surface;
this layer gradually detaches itself as the sea-water enters he space
next the cell-surface, and with the resulting distension the inequal-
ities disappear; after 20 to 25 minutes (at 20° to 22°) the membrane
is uniform and normal in appearance, though still very near the
egg-surface. The process may be characterized as secretory in
nature, and it appears to be dependent on a partial solution of the
superficial lipoid layer of the egg; this is indicated by its ready
production through the action of ‘the various fatty acids and fat-
solvents. The above temperature-relations appear to indicate,
in the case of production by warming, a dependence on some
enzyme action. Ifa simple solution of certain substances at higher
temperatures were the determining condition, the high tempera-
ture-coefhicient of acceleration, as well as the failure of tempera-
tures above 45° so to act, would be unintelligible. On the other
hand, the assumption of dependence on some process accelerated
by an enzyme with an optimum temperature of 38° to 40°, and
rapidly destroyed at 45°, would account for the above relations.
Certain hydrolytic cleavages may be concerned, possibly a saponi-
fication resulting in a partial solution of the surface layer; the
production of the same effect by the action of fat-solvents or alkalis
becomes readily intelligible on such an assumption.

An important significance has been ascribed by Loeb to the
process of membrane-production in sea-urchin eggs. After mem-
brane-formation, however induced, the condition of the egg is
altered in such a manner that relatively brief exposure to hyper-

386 Ralph 8. Lillie

tonic or hyperalkaline solutions is sufficient to produce normal
development.** Even without such after-treatment, eggs in which
membranes have been produced frequently cleave and under cer-
tain conditions may form blastulz; usually, however, such eggs
undergo breakdown or cytolysis within a few hours. Since this
aterare as well as the cleav age, 1S dependent on the presence of
free oxygen, the inference is drawn that in some manner, possibly
by removal of anticatalytic substances, membrane-formation leads
to an acceleration of oxidation processes in the egg; these if
properly directed i
fertilization—lead to cell division and development; otherwise
they result in the destruction of the egg. Membrane-formation
has thus an important significance in development.

My own observations on the starfish egg in some respects sup-
port this conclusion, though they can scarcely be said to do so
uniformly. ‘That the process of membrane-formation is not essen-
tial to cleavage has been known for some time; Loeb’s early studies
in artificial parthenogenesis supply instances of cleavage without
formation of fertilization membranes, and he cites other instances
in a later paper.* It is also possible artificially to suppress mem-
brane-formation without destroying the possibility of cleavage in
the following manner: Eggs were placed I5 minutes after renova
in KCN solution in sea-water, and remained here 20 OUI.
they were then washed in normal sea-water and warmed to 35°
for 70 seconds; these eggs formed no membranes although a con-
siderable proportion underwent irregular cleavage. On another
occasion the same suppression of membrane-formation without pre-
vention of cleavage was observed in eggs exposed to 34, KCN for
only two hours. Alchough cleavage is thus to a certain degree
independent of membrane-formation, nevertheless normal cleavage
and development certainly do appear to be facilitated by the separa-
tion of the membrane. In the above cited experiments develop-
ment stopped short at an early stage, and | have never found eggs
to develop to an advanced stage under this form of treatment
without the formation of amembrane. On the other hand, when-

18 Loeb: Joc. cit., also Archiv fiir die gesammte Physiologie, cxviii, pp. 181 and 572, 1907.
4 Loeb: University of California Publications, Physiology, vol. ii, p. 153, 1905.

Artificial Parthenogenests in Starfish Eges 387

ever mature eggs are treated in such a way as to form fertilization
membranes—whatever method is used—a certain proportion are
always found to undergo cleavage. Another observation that [
have frequently made appears to favor the idea that there is a cor-
relation between membrane-formation and the acceleration of oxi-
dation processes in the egg. I have found uniformly that the coagu-
lation and disintegration which follow when mature eggs are allowed
to remain for some hours in normal oxygen-containing sea-water,
occur much more rapidly in eggs that have formed membranes
than in those that remain without this structure. “Thus, warming
eggs during early maturation to 35° for 25 or 30 seconds induces
membrane-formation in a large proportion—usually about one-
half—but not in all of the eggs; practically all of the eggs so treated
die at an early stage; if theyare examined after 18 hours, those with
membranes are invariably found to be in an advanced state of
disintegration, the entire space enclosed by the membrane _ being
filled with a mass of loose granular detritus; those without mem-
branes, on the other hand, although coagulated and opaque, are
sull compact and undisintegrated. ‘The same contrast between
eggs with and without membranes in the rate and character of the
disintegration is seen when the membranes are formed by the
action of ether or a fatty acid. ‘This result, which I have found
with perfect uniformity throughout the present investigation,
shows that eggs which have formed membranes, yet without under-
going normal development, exhibit less resistance to the disinte-
grative action of the post-maturation oxidative changes than do
those lacking these formations. It is possible that the greater
cytoplasmic activity of the eggs with membranes (as shown by the
production of pseudopodia and the irregular cleavages and other
form-changes) may facilitate the disintegrative process; the effect
may also conceivably be dependent, at least in part, on simple
mechanical conditions: the change in the closely adhering surface-
layer of the unaltered egg, due to the removal of the membrane-
forming substance, would probably facilitate the action of any
disintegrative agency. One might suggest that the mechanical
resistance to surface-changes, including cleavage, is lessened by
the formation of a membrane, and that the significance of the

388 Ralph 8. Lillie

process in facilitating developmental changes may possibly lie
here.

The membrane is readily formed by brief exposure, during or
after the maturation stage, to the action of sea-water containing
xylol or ether; and such eggs show the typical irregular form-
changes and cleavages; | have however not yet obtained free-
swimming blastule from eggs thus treated. ‘Treatment for one
or two minutes with a solution of 3 cc. 34; acetic acid in 50 cc. sea-
water produces perfect membranes, and | have frequently obtained
a small proportion of swimming larvz from eggs so treated. The
effect must be regarded as due to the lipolytic action of the fatty
acid and not as a general effect of acidity (or increased concentra-
tion of hydrogen ions) since mineral acids—H,SO, and HNO,—
used similarly fail to produce the least sign of a membrane."

The ability of mature eggs to form membranes as a result of
momentary warming shows a certain periodical variation, as will be
shown in more detail later (cf. pp. 400, 403). In general the disso-
lution of the germinal vesicle is an important condition, although
immature eggs may form perfectly typical membranes under certain
conditions (p.407). Again,as already shown, membrane-formation
by heating becomes more difhcult after maturation is complete.
On the other hand, treatment with a fatty acid appears to produce
membranes with equal readiness at any time after maturation has
begun. Thus I have subjected successive portions of a single lot
of eggs to the action of the above acetic acid solution at 10 minute
intervals throughout the entire course of maturation (until the
separation of the second polar body) and again an hour later,
without finding any decided difference in effect at the different
periods; a small proportion of blastula was obtained in every one
of the ten experiments of the series except the first (treated 10
minutes after removal from animal). The largest proportion of
larvze was obtained from eggs treated previously to the separation

18 Compare Loeb: Joc. cit., and Dynamics of Living Matter, 1906, p. 172

16 Loeb (Joc. cit., cf. also Dynamics of Living Matter, p. 170) found the same difference between
fatty and mineral acids. Lefevre, however, finds that in Thalassema mineral acids produce membranes
with the same readiness as do fatty acids. Here apparently some other action than the directly lipolytic
is involved. Cf. Lefevre: Journal of Experimental Zodlogy, vol. iv, p. 106, 1907.

Artificial Parthenogenesis in Starfish Eggs 389

of the first polar body; still, no such well-defined periodicity was
found as with the experiments on the effects of warming (pp. 396,
et seq.) The appearances indicate a difference in the conditions
of the membrane-forming process—the acid acting by a simple
lipolytic action on the surface layer, while the effect of heating
depends on acceleration of an enzyme action, as already suggested.
Variations in the quantity or in the condition of the enzyme would
affect the results of warming without altering those due to the
simple action of a fat solvent.

Later I shall give some further observations on membrane-
formation in starfish eggs. The process certainly seems to be
correlated with a change in the developmental capabilities of the
egg. It does not however necessarily lead to an acceleration of
the oxidations in the egg, as is shown by the fact that immature
eggs in which membranes have been formed show no increased
disposition to undergo the typical oxidative coagulation or cyto-
lysis (p. 408); yet under certain conditions (after maturation has
begun) such an accelerated oxidation does seem to result and to
constitute an important condition of development, as already
indicated. ‘The experiments to be described later show, however,
that only a small part of the effects of momentary warming can
thus be accounted for. In the starfish egg, in fact, repression
rather than acceleration of oxidations seems to be an important
condition of the initiation of the developmental process, although
this latter once begun naturally requires free oxygen for its con-
tinuance (p. 413, et seq.)

B- Development of Eggs after Momentary Warming

Membrane-formation is followed after a more or less prolonged
interval by a series of form-changes in the egg; these under favor-
able conditions take the form of regular cleavage. It must be
regarded as significant that the most manifold and irregular
changes of form may occur, with all gradations between the pro-
trusion of pseudopodia and the assumption of various irregular
uncleaved forms or the production of irregular and unequal cleay-

ages and fragmentations on the one hand, and the normal process

390 Ralph S. Lillie

of typically regular and equal cleavage on the other. The irregu-
larities are extremely various, and it is difficult to assign any def-
nite conditions to the production of any particular kind. They
seem largely due to changes occurring in the cytoplasm independ-
ently of the nucleus; in other words, there is frequently a lack of
correlation between nuclear and cytoplasmic activities in the
warmed eggs; certain processes are initiated in both, sometimes
leading to nuclear division independently of cytoplasmic division,
at other times to the apparently independent assumption of irregular
forms on the part of the cytoplasm, with the production of irregular
pseudopodia, usually followed by subdivision of the cytoplasm into
unequal cleavage cells or still smaller enucleate fragments. Such
fragmentation is very typical of eggs that have been warmed
for too prolonged periods; the formation of small bead-like pro-
tuberances which then separate from the rest of the cell-body is an
especially frequent phenomenon. ‘These conditions as a rule
reach their height about three or four hours after warming, at a
time when the first cleavages usually begin to appear in regularly
dividing eggs.

The production of protoplasmic processes at times shows remark-
able peculiarities, particularly in eggs derived from animals late
in the season or otherwise unfavorable. ‘The proportion of irregu-
lar form-changes is also greater in eggs warmed after maturation
is complete (p. 402). A slightly prolonged warming often leads
to the production of numerous long slender close-set pseudo-
podia of clear protoplasm, of a uniform length sometimes equal
to that of the egg-radius, imparting a prickly or radiating appear-
ance to the entire structure; irregular fusions may take place between
these processes as in the pseudopodia of Foraminifera.‘7 These
cytoplasmic activities seem to have little directly to do with nuclear
influence; separated enucleate portions of protoplasm may also
undergo irregular form-change or subdivide still further. Other
instances of specific change of form in enucleate portions of eggs
have been described by several observers. It seems clear that the

17 Such conditions seem frequent in abnormally developing parthenogenetic eggs; compare especially
the accounts of F. R. Lillie for Chetopterus, /oc. cit., p. 487; also of Lefevre for Thalassema, /oc. cit.,

p- 109.

Artificial Parthenogenesis in Starfish Eggs 391

cytoplasm possesses a certain formative capacity of its own;'8 this
under the above abnormal conditions may g give rise to structures
having very definite peculiarities, but quite foreign to the normal
development.

Under favorable conditions a large proportion of eggs undergo
regular cleavage and development. ‘The following series rable
IL) will illustrate; he eggs (all from a single lot) were exposed to
temperatures of 35°, 36°, 37° and 38°, for varying brief periods
during the early maturation period (between 20 and 45 minutes
after removal from the animal, before separation of the first
polar body). The susceptibility varies somewhat within this
period; but, as will be shown later, warming may produce develop-
ment at any time between the dissolution of the germinal vesicle
and the formation of the first polar body (after which time it
becomes increasingly difficult to incite development by this means).
Within at least the greater part of the period of exposure covered
by this series the susceptibility to development by warming varies
relatively slightly, and the condition of the eggs may be regarded
as essentially uniform throughout. Later experiments will be
described in which the variation in susceptibility at different
periods during maturation is itself made the subject of study (cf. pp.
396, et seq.)

Eggs from the same lot were treated in a precisely similar
manner on the afternoon of the same day, from 2.36 to 3.06 p.m.
All had matured in the typical manner. The result was quite
different. Membrane-formation was less uniform and required
a more prolonged exposure to the respective temperatures, and
although in favorable experiments a considerable proportion of
eggs underwent cleavage, mostly irregular, not a single swimming
larva was obtained. ‘This kind of experience has been aoe
Ihave never succeeded, afterthe completion of maturation, in bring-
ing unfertilized eggs to the free-swimming stage. “The eggs invari-
ably either fail to cleave, or cleave more or less irregularly, usually
after undergoing rregular form-changes, and die at an early stage.

18 Compare Wilson’s account of the phenomena in the isolated enucleated polar lobe of Dentalium;
cf. also the references in his paper to analogous phenomena in echinoderm eggs. Wilson: Journal of
Experimental ZoGlogy, vol. 1, p. §3, 1904.

Bs Ralph S. Lillte

TABLE II

August 8, 1906. Eggs were removed from the animal at 10:15 a.m., and treated as follows: Tempera-

ture of sea-water in the dishes, 23°

Interval
ntact Temperature and

removal from

blastule obtained
24 35 60 | All form membranes. Cleavage largely regular; blast-
ule more numerous than in Experiment 3
All form membranes. Cleavage more regular and rapid

25) 35,5 792
| than in Experiment 4; good proportion gastrule

27 35 80 | Similar to 5; good proportion blastule and gastrule

29 | 36 15 | Practically no membranes (one seen). No cleavage

29.5 35 20 | Allform membranes. Mostly irregular cleavage. No larve

30 | 36-30 Similar to 8

31 36 40 | More favorable; large proportion regular cleavages and a
| fair number of blastule and gastrule

32 36 50 Somewhat more favorable than 10; a considerable number

of blastule and gastrule

20 |

36 a7; 10 A few membranes formed; no cleavage found

37 37 15 | Most form membranes: cleavage mostly irregular; no
blastule obtained
38 37 20 All form membranes; mostly irregular cleavages, a few

regular; no larve found

39 | 37 30 More favorable than 14; good proportion regular cleavages;
large number blastule and gastrule obtained, and a few
good Bipinnarie

40 37 40 Fewer regular cleavages than in 15; a fair number of larve
obtained

41 38 5 Hardly any membranes (2 or 3 seen). No cleavages found

41 38 10 Almost all form membranes. Cleavage irregular or incom-

plete. No larve
42 38 15 All form membranes and cleavage is less irregular than in
18. Some regular cleavages, and a few blastule and
gastrule obtained
43 38 20 Large proportion of irregular cleavages and a fair propor-
tion give swimming blastule and gastrule; a few reach

Bipinnaria stage

Artificial Parthenogenesis in Starfish Eggs 393

The time of early maturation (before the separation of the first
polar body),is apparently a critical period for the production of this
type of parthenogenetic development. The same has been found
true by Delage."

A similar series of Spa vata on July 24, 1907, with the three
temperatures 35°, 30° and 37° and a somewhat different range of
exposures gave in general the same result, summarized in Table

TET.

TABLE IT

Fuly 24,1907. Eggs were removed at 2:15 p.m., and treated as follows

Interval after | Temperature and |
removal exposure eS
| minutes (ca.) | deg. sec.
To] 30 | 35 60 | Good proportion of regular cleavages, and fair number
blastula and gastrule
zy 31 35 7° | Cleavage more rapid and regular than in 1; large number
| active larva obtained
yi 33 ie te) In general like Experiment 2: somewhat less favorable;
| numerous larva
4 34 | 35 go | Smaller proportion of regular cleavages and fewer larve
than in Experiments 2 and 3
5 37 | 36 50 All form membranes, but cleavage is mostly irregular; no
larve
6 38 36 40 Larger proportion cleavages than in 5, largely regular. No
| larvee
7 40 36 50 Fair proportion of regular cleavages, fair number of blas-
tule and gastrule obtained
8 | 42 | 36 = 60 Cleavage less regular and slower than in 7; good many
| | blastule and gastrule
y | 45 37 20 All maturing eggs form membranes, relatively few cleav-
ages; no larve obtained
10 46 37 30 Large proportion of regular cleavages; fair number of
blastule and gastrule obtained
II 47 37 40 Fewer cleavages, slower and less regular than in Experi-
| ment 10; eggs mostly adopt irregular shapes without
cleaving: a few larve
12 | 48 37 50 Eggs form membranes and adopt irregular shapes with
| slender pseudopodia; no cleavages found. No larve

19 Delage: Joc. cit.

394 Ralph S. Lillte

From the above experiments it appears that the optimum time
of exposure to the high temperatures is shorter the higher the tem-
perature employed. In the above two series the best results were
obtained at 35° with 70 seconds exposure, at 36° with 40 or 50
seconds, at 37° with 30 seconds, and at 38° with 20 seconds. In
general the same relative favorability of different periods of expo-
sure at different temperatures was found in several other similar
series of experiments. The decrease in the optimum time of
exposure with a given increase in the temperature is somewhat
surprisingly rapid, the difference of three degrees between 35° and
38° reducing the optimum exposure from 70 to about 20 seconds.
If the process in which the initiation of development depends is
a purely chemical one, this would indicate an extraordinarily high
temperature-coefhicient of acceleration. The conditions in a
heterogeneous system like protoplasm must, however, be recog-
nized as peculiar; rise of temperature, in addition to accelerating
the specific chemical processes (usually about threefold for a rise of
10°), has a certain effect (which under some conditions may be very
considerable) in altering the surface of interaction between the
colloidal bodies concerned and the other chemical substances
reacting with them; increased subdivision of the colloidal particles
following a rise of temperature would in itself accelerate the reac-
tion; and the total acceleration would be measured by the product
of this increase in the surface of interaction into the specific acceler-
ation of the process itself through the rise of temperature; this com-
pound value might exceed many times (as apparently in the case
under consideration) the simple temperature-coefhicient of acceler-
ation of the purely chemical process. “The results with membrane-
production also indicate a very high temperature-coefhcient. So
far as regards development I have as yet made no special endeavor
to determine the optimum periods of exposure at temperatures
above and below those cited. ‘The favorable periods for tempera-
tures of 39° and 40° would undoubtedly be found very short, while
at 34° and 33° exposures would be more prolonged. “Temperatures
so low as 30° would in all likelihood be found effective with sufh-
cient time of exposure, as Delage’s*® results indicate.

20 Delage: Joc. cit., p. 307+

Artificial Partheno genesis in Starfish Eggs 395

The different temperatures do not however seem equally favor-
able, and more larve are obtained at 35° and 36° than at 37° and
38°; in other words, with the higher temperatures warming seems
more likely to produce abnormal results. My experience has been
that temperatures of 35°, acting for somewhat longer than one
minute, have usually given the best results. ‘This is illustrated by
the foregoing tables and again by the following four experiments.
In this series eggs from a single animal were exposed to 35° for
60, 70, 80 and go seconds, respectively, at a period of 30 to 35
minutes after removal from the animal. The results were as
follows:

1 | 35° 60s. | Agood proportion reach gastrula stage and a few form early Bipinnarie
2 | 35° 70s. | A large proportion reach advanced gastrula stage and a considerable number
form early Bipinnarie
3 35° 80s. | Somewhat less favorable than Experiment 2; a considerable number form
| advanced gastrula, and a few early Bipinnarie
4 35° gos. | Fewer larve obtained than in the above three experiments and none reach early

| Bipinnaria stage

An exposure of 70 seconds to 35° again proves most favorable.
In all of the following experiments on the determination of the
period of greatest susceptibility to this treatment I have accordingly
employed uniformly an exposure to 35° for 70 seconds; such treat-
ment if applied at a favorable period (best at some little time before
the separation of the first polar body) almost invariably results in
the production of a good and sometimes a high proportion of
larva. There appears however to be some variation in the opti-
mum period of exposure to a given temperature in eggs from differ-
ent animals and at different seasons of the year. “Thus on Septem-
ber 6, 1906, eggs were treated as follows during early maturation
(17 to 37 minutes after removal): 35° for 70, 75, 80 and 85 seconds;
36° for 40, 45, 50, 55 and 60 seconds; 37° for 20, 25, 30 and 35
seconds. Cleavage was most nearly normal and a certain rather
small proportion of larva was obtained with 35° for 85 seconds, 36°
for 50, 55 and 60 seconds (the last best), and 37° for 30 and 35
seconds (both about equally good). With the other exposures the
cleavage was slower and less regular and no swimming larve

396 Ralph 8. Lillie

resulted. Another portion of the same eggs was similarly treated
in the afternoon about three hours after completion of maturation;
a large proportion failed to form membranes, cleavage was either
irregular or failed altogether, and not a single larva resulted.
This series was less favorable than those tabulated above, and the
optimum exposures were considerably more prolonged. The dif-
ference in the time of year may be a factor of importance; this,
however, can only be determined by further experiment. On the
whole, when normal eggs are used a given temperature has a
quite well defined optimum period of exposure which can be de-
termined with considerable accuracy. Since the temperature-
coefficient of acceleration of a given process may afford valuable
indications as to its essential nature, a more exact redetermi-
nation of the optimal periods of exposure through a greater
range of temperatures may yield valuable results. I hope at
some future time to make more extended and exact determina-
tions of the above and similar relations.

Susceptibility to Warming at Different Periods During Maturation

The foregoing experiments had shown that momentary warming
has a favorable action in inciting parthenogenetic development
during, but not after, the period of maturation. It remained to
determine more precisely the limits of the period of susceptibility
to this form of treatment, and the variation in favorability within
this period.

For this purpose in each of the series of experiments tabulated
in Table IV the eggs of a single female were employed; successive
portions of these were warmed to 35° for 70 seconds, beginning
about five minutes after removal (at a time when the germinal
vesicle had undergone no visible alteration), and thereafter at
regular five minute intervals until the separation of the first polar
body had taken place. The condition of the eggs at the time
of warming was observed in each case in a “control” portion
kept under the microscope throughout the entire period of the
series. With good eggs from a single female the maturation proc-
ess progresses with almost uniform velocity in all eggs; the numer-
ous eggs of each portion may thus be considered practically uni-

Artificial Parthenogenesis in Starfish Eggs 397

form so far as regards the stage at which the treatment was applied;
all portions were treated exactly alike. The results indicate the
existence of a fairly well defined period of maximum susceptibility,
lasting for a certain period preceding the completion of the first

maturation division.
favorable.

Thereafter conditions become rapidly less

The results of three satisfactory series of experiments are
summarized in the following table:

TABLE IV

The temperature of exposure was 35° and the time 70 seconds in all cases.

The condition of the eggs at

the time of exposure in each experiment is indicated by the italicized portion enclosed in parentheses.

The time itself (interval after removal of eggs from animal) is given in the second column

| tinct in nearly all)
| larger proportion form
|

larve than in Experiment
3,and reachmore advanced

stage (gastrule formed)

about one-third of the
eggs; rest remain imma-
ture) Larger propor-
tion cleavages than in
Experiment 3; no larve

are maturing, vesicle in-
distinct) A fair propor-
tion develop as in Ex-

periment 3

Ralph S. Lillie

TABLE [V—Continued

No.

Time after| RESULTS

removal

minutes Series I Series IT Series III
(ca.) July 31, 1907 August 7, 1907 August 12, 1907

25 (Vesicle almost disappeared (As in Experiment 4; only (Germinal vesicle disap-

in nearlyall) Morefav- one-third or so maturing) _ pearing in ca. one-half of

orablethanExperiment4; Largerproportioncleave the eggs) Large num-

larger proportion larve than in 4; fair number ber good blastule and
blastule gastrule formed

30 (Vesicle barely distinguish (About one-thirdofthe eggs (About one-half of the eggs
able in most) Similar fo} maturing) Cleavage as) maturing) Larger pro-
Experiment 5 | inExperiment 5;nolar- portion of Jarve than in

ve found 5

35 (Vesicle indistinct in most) (Like 6: one-third matur-| (Like6) More favorable
| Numerous larve formed; ing) Cleavage rather than 6; large proportion

more favorable than Ex-- more regular than in of good gastrule formed
periment 6 | 65 fair proportion form
blastule

40 (Region of germinal vesicle) (One-third mature; a (As before; first polar spin-
| almost indistinguishable) polar spindle visible as dle visible in about one-
Larger proportion cleave clear area at surface of half of the eggs) Large
and more regularly than egg) A few blastule| proportion of eggs form
in 7; next morning most) formed; less favorable gastrule as in 7; rather
eggs in blastula or early) than7 | more favorable
gastrula stage

45 (Like 8) Most eggs form (As in 8) Less favorable) (Like 8) Cleavage slower

vigorous gastrule by next’ than 8; no larve and fewer larve formed
morning; some reach Bi- than in 8; still very fa-
pinnaria stage later. Very vorable

|
|
| favorable culture

° Vesicle quite invisible in (Like8 andg) Unfavor- | (Like 8; polar bodies not
5 q Shs
practically all eggs) Less, able; eggs take irregular) yet separated) Less fa-

favorable than 9; till as) shapes; no larve vorable; cleavage more
large proportion form vig- irregular and few form
orous larve a few of which larve

reach early Bipinnaria)
stage

Artificial Parthenogenesis in Starfish Eggs 399

TABLE IV—Continued

Time after RESULTS
No. | removal |——— : =
minutes Series I Series II Series HT
(ca.) July 31, 1907 August 7, 1907 August 12, 1907
II 55 (Like1o) Numerousactive) (Polar bodies not yet sepa- (No polar bodies as yet)
| larve, some reaching) rated) Cleavage irregu-) Cleavage slow and more
early Bipinnaria | lar; no larve formed irregular than in 10; not
a single larva
|
12 60 (Polar bodies not yet sepa-| (Polar bodies beginning to, (Polar bodies beginning to
rated) Decidedly fewer| separate) Cleavage ir- separate) Cleavage ir-
larve and less activethan| regular; no larve | regular; no larve
in 11 |
|
13 65 First polar bodies have sep-| (First polar bodies sepa-| (Polar bodies separated in
arated in many eggs)| rated in the mature eggs.. maturing eggs) No de-
Cleavage much retarded) 1. e., one-third of whole) velopment
and only afew small ir-/ Similar to 12
regular blastule obtained |
14 17 (Polar bodies separated), (Like 13) Like 13; no, (Like 13) No develop-
Cleavage delayed and ir-) larve ment
regular; very few larvae;
feeble and abnormal

In the above three series a large number of eggs reached the
larval stage, Series I being especially favorable. In two other
similarly conducted series the eggs proved inferior, only about 10
per cent undergoing maturation. In the first of these series
August 10, 1907, larva were obtained only from eggs warmed at
periods corresponding to Nos. 5 and 8 of the above series; the
second, August 13, proved somewhat more favorable, blastula
resulting from eggs exposed at periods corresponding to Nos.
Asis Os 7> 8 and g above with the optimum at Nos. 6 and 7. The
suppression of maturation in eggs warmed within 5 to 10 minutes
after removal also resulted in Shock series. Three other similar
series—July 29, July 30 and August 3
suppression of maturation in eggs heated directly after removal.
The eggs in these series were inferior and no larve were obtained;

400 Ralph S. Lillie

but in the best experiment, that of July 29, the largest proportion
of regular cleavages—and in general the most favorable condi-
tions—was found in eggs warmed at stages corresponding to
Nos. 8, 9 and 10 of the above table.

In the series tabulated above the following chief uniformities
are apparent: (1) Warming shortly after removal (within 5 to 10
minutes, before the germinal vesicle has undergone any apparent
change) has the effect of completely preventing maturation; the
germinal vesicle remains intact and the egg remains permanently
in this condition until disintegration sets in. Such eggs behave in
the same manner as do eggs that fail to mature from any other
cause—they remain clear and apparently unaltered at a time
when mature eggs have undergone complete coagulation and dis-
integration. (2) Warming at any time after the germinal vesicle
membrane has begun to dissolve and before the separation of the
first polar body may lead to development and production of larve;
the proportion of eggs that develop is at first small, but increases
rapidly; in general the conditions for development steadily improve
until a certain stage is reached—about 40 to 45 minutes after
removal at normal summer temperature; at this time the suscepti-
bility of the eggs is greatest and momentary warming is followed
by development and the production of active larve in a large pro-
portion. ‘Thereafter the susceptibility rapidly declines; and at the
time of separation of the first polar body warming results chiefly in
abnormal development and larve are rarely obtained.

Warming in later periods is still less favorable. In the following
two series eggs were treated as above at 10 minute intervals until
after the formation of both polar bodies, and after this less fre-
quently until about five hours after removal. Both series proved
favorable and showed good agreement; larva were most numerous
from eggs warmed 10 to 15 minutes before the separation of the
first polar body; at the time of separation few or none were
obtained, and thereafter conditions became progressively more
unfavorable with lapse of time. After both polar bodies had
separated the eggs not infrequently failed altogether to cleave
or even to produce membranes—a result which agrees with those
of the earlier experiments already cited.

Artificial Parthenogenesis in Starfish Eggs 401

TABLE V

Series I, Aug. 18, 1907. . Eggs were removed from Series II, Aug. 20, 1907. Eggs were removed at
animal at 11:12 a.m., and warmed to 35° for 70 9:30 a.m., and warmed to 35° for 70 seconds after

seconds at following intervals after removal. the intervals indicated

Interval RESULT | Interval RESULT
I Ism. (Germinal vesicle beginning 7m. (Germinal vseicle unchanged)
to show signs of dissolu-| Practically all eggs remain
tion) A few eggs reach 1 in immature state; no de-
blastula stage |} velopment
|
3 25m (Germinal vesicle indistinct | 17m (A few eggs show beginning
| in large proportion of eggs) I maturation) A few feeble
Large number form blas- | blastule formed
tule and gastrula |
3 35m (Germinal vesicle invisible 27 m. (A small proportion of eggs
in most eggs) Good pro- maturing) Considerable
portion blastula and gas- | number of blastula ob-
trule tained
4 45m | (First maturation spindle at 37 m. (Only 5 to 10 per cent eggs
surface; no polar bodies | maturing) Fair number
separated) Good propor- | of blastule and gastrule
tion blastule and gastru- |
le |
5 55m. | (First polar bodies separat- | 47 m. (First maturation spindle at
ing) A good many eggs surface of maturing eggs)
seem to have cleaved reg- More favorable than Ex-
ularly but no larve were periment 4; good propor-
obtained portion form blastule and
| gastrule
|
6 | th.gm. | (Al maturing eggs have first 57 m. (Polar bodies not yet sepa-
| polar bodies) Mostly ir- rated) A fair proportion
| | regular cleavages and frag- | of larve; rather fewer than
| | mentation; no larve | in Experiment 5
| | }
7| th.1gm. | (Second polar bodies not yet i 1h.7m. | (First polar bodies beginning
| separated) After 8 hours | | 10 separate in maturing
most eggs still uncleaved; | | eggs) A few larva; less
a few irregular cleavages; | favorable than 6
no further development

Artificial Parthenogenesis in Starfish Eggs 403

TABLE V—Continued

Interval RESULT Interval RESULT
rsa subs The same as above; eggs do|| 15 | 4h. 44m _ | After 4 h. 15 m. almost all
not form membranes and mature eggs found coarsely
coagulate within 5 hours coagulated and in process

of disintegration, mostly
(but not all) without mem-
branes

|| 16} 5h. 35 m. | After 34 hours mature eggs
| are in process of coagula-
tion; only a few have mem-
branes

I find no record in my notes of the condition of the membranes in Experiments 12 to 14 of Series II.
In this series membrane-formation is not so completely absent as in Series I in the stages succeeding
the completion of maturation; but presumably the proportion of eggs without membranes increased
steadily from Experiment 11 to Experiment 16 where only a few were formed.

In both of the above series the eggs show a progressively increas-
ing inability to respond to momentary warming after the maturation
process is complete. The proportion of eggs that fail to form mem-
branes also increases with the lapse of time in the post-maturation
stages. Cleavage becomes irregular or fails altogether; the
curious result also appears in the experiments made some time
after the complete separation of the second polar body that the
tendency to coagulation, typical of mature unfertilized eggs, is
markedly accelerated. ‘This effect is conspicuous in eggs warmed
at a stage of four or five hours after removal, as seen in both of the
above series; the coagulative process is well advanced in such eggs
within three or four hours after warming; while in mature eggs not
exposed to this treatment, or warmed at anearlier stage, coagulation
does not become evident until some hours later. The process, as
Loeb has shown, is oxidative in nature; warming in post-matura-
tion stages has thus the effect of accelerating oxidations leading
to a coagulative disintegration of the egg-substance. An earlier
experiment performed with another object in view shows the same
result: Eggs were removed at 11:30 a.m. August 5, 1907; about

404 Ralph S. Lillie

3 h. 45 m. later they were warmed to 35° for the periods indi-
cated.

I 35° 60 s. (3:15 p.m.) | 3h. later (6:15) only a few eggs have membranes; most are unaltered;
no cleavage

2 35° 65 s. (3:16 p.m.) | At 6:20 condition similar to Experiment 1; only a few eggs have mem-
branes; these are dead and in process of coagulation

s. (3:18 p.m.) | At 6:21 the mature eggs are all opaque and coagulated; only a few

ww
we
nm

°
x
°

have membranes; these uniformly show the greatest disintegration
75 Ss. (3:20 p.m.) | At 6:21 condition of eggs similar to Experiment 3

5 | 35° 80s. (3:22 p.m.) | Similar to Experiments 3 and 4

In the unfertilized unwarmed control at 6:23 p.m. the eggs
remained quite uncoagulated; warming has thus hastened the
coagulative change in the mature eggs, and especially in those with
membranes, which uniformly showed the most advanced disinte-
gration,

What are the conditions of this varying susceptibility to the
above form of treatment at these different periods in the life of
the unfertilized egg? One event, occurring shortly after the
removal of the egg from the animal to normal oxygen-containing
sea-water, seems of fundamental significance, viz: the dissolution
of the membrane of the immature egg-nucleus or germinal vesicle.
This event naturally must precede the maturation divisions that
follow; but quite apart from this it seems to form the condition of
a profound change in the properties of the egg-cytoplasm. Delage”!
has found that enucleate egg-fragments of Asterias are insuscepti-
ble to fertilization before the germinal vesicle has undergone visible
change; but that very soon after its membrane has begun to show
indication of dissolution, merogonic fertilization first becomes
possible; a little later, when the membrane has become invisible—
although the area of the former germinal vesicle may still be seen,
often with nucleolus Waar the: fragments of cy toplasm are com-
pletely and readily fertilizable. Tiese observations demonstrate
that the essential feature of maturation, so far as the cytoplasm is
concerned, is not the separation of the polar bodies, but simply the
removal of the barrier between the nuclear and the cytoplasmic

21 Delage: Archives de zoologie expérimentale et générale, Sér. IIT, T. 9. p. 285, 1901.

Artificial Parthenogenesis in Starfish Eggs 405

areas; they also show that the nuclear membrane acts as a semi-
permeable membrane with reference to certain substances con-
tained within it. The critical event, therefore, which conditions
this remarkable change in the properties of the cytoplasm is,
according to Delage, the passage of certain nuclear constituents (suc
nucléaire) into the cytoplasmic area (Joc. cit., p. 289). These
substances he suggests may either change the osmotic pressure
of the cytoplasm, or may influence the rate of oxidations, or may be
of the nature of particular electrolytes or enzymes. He was unable
to produce by artificial means any developmental changes in
such egg-fragments.

The precise nature of the change induced in the egg-cytoplasm
in consequence of the dissolution of the germinal vesicle is as yet
unknown. The fact that the egg, if not fertilized within a few
hours, readily undergoes an oxidative change involving a coagula-
tion of the cytoplasmic colloids seems to point to an acceleration
of oxidations in that region—due possibly, as lately suggested
in an interesting paper by Mathews,” to the liberation of oxidases
formerly confined to the nuclear area; if oxidases are of nuclear
origin, as certain facts seem to indicate,” such a consequence
would naturally follow; the periodic dissolution of the nuclear
membrane in mitotic cell-division would thus have the significance
of providing for the distribution of the oxidases (synthesized in the
nucleus) throughout the cytoplasmic area; this would naturally
result in a periodic acceleration of oxidation processes in the cell.
Lyon” has in fact shown that the production of carbon dioxide by
the dividing egg follows a rhythm parallel with that of the nuclear
division; and Loeb* has connected these oxidations with the syn-
thesis of nucleins from the compounds of the cell-protoplasm—
a process which is likewise characterized by a rhythm parallel with
that of the mitotic process.

This general interpretation, though suggested by quite different

= Mathews: American Journal of Physiology, vol. xviii, p. 94, 1907.

* Cf. the references in my paper On the Oxidative Properties of the Cell-nucleus, in American
Journal of Physiology, vol. vii, p. 412, 1902.

* Lyon: American Journal of Physiology, vol. xi, p. 52, 1904.

% Loeb: Biochemische Zeitschrift, vol. i, p. 183, and vol. ii, p. 34, 1906.

406 Ralph S. Lillie

considerations, is in striking agreement with the view propounded
by Conklin” some years ago in his studies of karyokinesis in the
Crepidula egg. Some of his conclusions on the physiology of this
process should be quoted. ‘‘The nuclear membrane appears to
permit the passage of materials inward but not outward during the
resting period; whereas the escape of nuclear material into the cell
is brought about by the disappearance of the nuclear membrane
during karyokinesis.”” In Crepidula there can be demonstrated
cytologically “a very extensive interchange of material between
the nucleus and the cytoplasm;” “a large part of that most
characteristic nuclear substance, the chromatin, passes into the
cytoplasm in the form of oxychromatin during every cell-cycle,
while a relatively small part is reserved for the purpose of repro-
ducing the daughter-nuclei.” This passage of nuclear material
Gewreele aucleo- -proteid in nature) into the cytoplasm is re-
garded as a fundamentally important condition of the subsequent
game undergone by the latter. These phenomena appear to be
characteristic of mitosis in general and essentially similar conditions
have been described for a penance of cells. In the starfish egg by far
the greater part of the chromatin is set free in the cytoplasm during
the first maturation division.?’? In Chetopterus also the greater
part of the germinal vesicle consists of a “residual substance”
which is set free in the cytoplasm at the first maturation-division
and plays an important part in the future development.’ It is
natural, in view of the probable nucleo-proteid nature of at least
certain enzymes, to regard the above “‘oxychromatin” or “residual
material’? as consisting—at least in part—of the ferments con-
cerned in the chemical processes—largely oxidative in their nature
as shown clearly by the conditions in the starfish-egg—that deter-
mine the later characteristic changes in the cytoplasm. The
ascertained cytological facts are thus in essential harmony with
the above hypothesis.

Whether the change in the cytoplasm depends primarily on
increased oxidations or on other conditions is scarcely decided as

% Journal of the Academy of Natural Sciences of Philadelphia, second series, vol. xii, pt. 1, 1902.
°7 Wilson and Mathews: Journal of Morphology, vol. x, p. 334, 1895.

28 Cf. F. R. Lillie: Journal of Experimental Zodlogy, vol. iii, p. 153, 1906.

Artificial Parthenogenesis in Starfish Eggs 407

yet. My own observations agree with Delage’s and those of
later observers in indicating that the dissolution of the nuclear
membrane is in some way associated with a well-defined alter-
ation in the capacity of the egg for further development. Mo-
mentary warming previously to this event not only fails to result
later in cleavage, but it has the effect of completely preventing
the change in question and with it the entire maturation-proc-
ess. On the other hand, as already seen, the same treatment
applied at any time after the beginning of the maturation-changes
(until the separation of the first polar body) may lead to develop-
ment and the production of larvae. The properties of the cyto-
plasm thus must undergo a profound change the nature of which
remains to be determined.

One normal sequence of the dissolution of the germinal vesicle
is a change in the reaction of the egg-cytoplasm toward membrane
forming agencies. Membrane-formation now promptly follows
warming, or the entrance of a spermatozoon, or the momentary
action of a fatty acid or fat solvent; while in the immature egg this
structure is usually not formed under these conditions.** Yet, al-
though as a rule eggs that remain permanently immature as above
do not form fertilization membranes on warming, this is by no
means invariably the case. I have recorded numerous instances
in which momentary warming has produced perfectly normal
membranes in immature unfertilized eggs. In general such eggs
belonged to lots that were unfavorable as regards capacity for
development, so that the membrane-production may be considered
as evidence of a certain abnormality. The following instance will
illustrate: in the series of August 2, 1907, cited above, in which
eggs were warmed at 5-minute intervals (until the separation of
the first polar body in those maturing), the majority failed to
mature, and the developing mature eggs in no case reached the
free swimming stage. In this series most of the permanently im-
mature eggs, after subjection to the momentary warming process,
formed quite typical uniform membranes indistinguishable from
those found in fertilized mature eggs; this was especially true of

29 Cf. Loeb: University of California Publications, Physiology, vol. ii, p. 150, 1905.

408 Ralph S. Lillie

those warmed at periods of 10 to 15 minutes after removal; after
an hour (at which time most mature eggs had formed polar bodies)
the proportion of immature eggs that formed membranes had
declined considerably, and at later stages only a few were formed.
This is not in the least an isolated observation, but 1s fairly typ-
ical of what I have frequently observed; the ability of immature
eggs to form membranes seems in general ‘best marked shortly
after removal, and diminishes after an hour or more in sea-water.
An observation made on the same lot of eggs showed that sperma-
tozoa may also induce membrane-formation in immature eggs;
sperm was added at 1 h. 25 m. after removal; the next morning
a fair proportion of the mature eggs had formed larva; and nearly
all of the immature eggs showed perfectly typical sharply defined
fertilization-membranes; otherwise these eggs remained unchanged.
Spermatozoa are known to enter immature starfish-eggs,*° but
typically to produce no membranes. Under certain conditions
however, not definitely understood (eggs “over-ripe”’ or otherwise
not quite normal), membranes may be formed as just seen, either
by spermatozoa or through an artificial agency. The explanation
may be as follows: normally the possibility of membrane-forma-
tion depends on the passage of certain substances from the nucleus
to the cytoplasm, since the beginning of maturation is a prerequi-
site; in the above eggs however the permeability of the germinal
vesicle membrane is abnormal, so that the substances necessary
to the membrane-formation, which ordinarily are unable to tra-
verse the nuclear membrane, are now able to effect this passage
and to enter the cytoplasm. ‘The latter then reacts to heat or the
entrance of the spermatozo6on by forming a membrane in the man-
ner characteristic of mature eggs.

It is interesting also to note that such immature eggs show no
other change in their properties; they remain clear and unaltered
for prolonged periods and show no greater tendency to disinte-
grate than do normal immature eggs—a fact apparently contra-
dictory of Loeb’s view that the separation of the membrane in-
volves an acceleration of oxidative processes in the egg. In mature

39 Wilson and Mathews: Journal of Morphology, x, p. 319, 1895.

Artificial Parthenogenesis in Starfish Eggs 409
eggs, however, there is an obvious difference in the conditions;
the entire contents of the germinal vesicle—not only those sub-
stances that can pass the nuclear membrane—have become mingled
with the cytoplasm; and in fact mature eggs differ from immature
eggs in undergoing the typical disintegration much more rapidly
after forming membranes, as shown above. It is quite possible
that for the oxidations concerned in the post-maturative disinte-
gration of the cytoplasm there is needed the presence of specific
papers: derived from the nucleus—e. g., oxidases, or enzymes or
proferments of some other kind, or certain activ ators—and that
these substances merely find better conditions for their activity
after the separation of the fertilization-membrane than before.
In their absence membrane-formation would in itself effect no
esential change in the condition of the cytoplasm. Membrane-
formation alone is thus quite ineffective—unless accompanied by
certain other and independent changes—in accelerating oxidations
in the egg-cytoplasm.

The effect of momentary warming in preventing the dissolu-
tion of the germinal vesicle is curious and difficult to explain.
The process itself, as shown by Loeb" some years ago, depends on
oxidations, since it 1s prevented by acidulation of the sea-water
or by depriving the eggs of free oxygen. One of his observations
seem analogous to Tet one under discussion: exposure of unripe egg
even temporarily (as for 15 minutes), to acidulated sea-water
(100 cc. sea-water + 5 cc. *; HNO,) prevented the eggs from matur-
ing after retransfer to normal sea-water. An oxidative process
therefore which normally leads to the dissolution of the nuclear
membrane within a few minutes after the eggs are laid, 1f checked
before that time is ordinarily not resumed and the eggs remain
immature. But why should temporary warming at this stage
produce a similar result? The expectation would be that by
such treatment the oxidations, as well as the other chemical proc-
esses in the egg, would be accelerated, and that a process like
maturation, dependent on oxidations, would be furthered rather
than prevented. Evidently warming, during the brief period that

* Loeb: Archiv fiir die gesammte Physiologie, xciil, p. 59, 1902.

410 Ralph §. Lillie

normally precedes the solution of the nuclear membrane of the
immature egg, in some manner inhibits the oxidations on which
this change depends. Just why this effect should result remains
for the present obscure; possibly several distinct chemical proc-
esses are concerned, having different coefficients of acceleration
by rise of temperature; at the higher temperature the available
oxygen may enter into a quite different reaction from that on
anal the maturation-change depends; the latter would then be
prevented through a deficiency of available oxygen. Ovxidations
in one set of processes may easily involve reductions in another
if the supply of free oxygen is limited. What is remarkable is
that maturation is prevented permanently by warming at this
stage. Warming after the germinal vesicle has broken down has
no effect on the course of maturation, the polar bodies forming in
the usual manner; and after this process is complete the eggs, as
already seen, may proceed to cleave and develop without fertiliza-
tion. Apparently conditions unfavorable to maturation produce
a permanent prevention of the process only if they act during the
brief period immediately following the deposition ‘of the eggs; this
is for some season a critical stage, and if the maturation process
is not then begun it fails altogether. In harmony with this inter-
pretation is the well known fact that starfish eggs which show no
signs of maturing by twenty minutes or so alter removal from the
neta to Roane sea-water remain immature permanently.

The effects of momentary warming at stages succeeding the disso-
lution of the germinal vesicle vary, as just shown, according to the
exact period at which the treatment is applied. As already seen,
membrane-formation and development may result from warming
very soon after the vesicle begins to lose its distinct outline. The
conditions are at first unfav orable, only a small proportion of eggs
forming membranes, and still fewer dev eloping to a free-swimming
stage. In general, as indicated by Table IV, the proportion of
favorably developing eggs shows a progressive increase until an
optimum stage 1s reached—usually about 15 or 20 minutes before
the separation of the first polar body; warming at the time of
separation of this polar body rarely results in larvae, and in later
stages the conditions become steadily less favorable with lapse
of time.

Artificial Parthenogenesis in Starjish Eges 411

The conditions of this change of susceptibility are at present
unknown. I have endeavored to determine if a similar variability
exists in respect to fertilization by spermatozoa; and the result
has appeared that although normal fertilization is possible through-
out a far greater period in the history of the egg (namely, at any
time after maturation has begun until several hours after its
completion) a very similar variation in the degree of susceptibility
to the fertilizing influence does in fact exist. Conditions for fertili-
zation by spermatozoa are at their best during the maturation period,
at or about the time of separation of the first polar body; and later
they become less favorable. There is thus a certain parallelism
between the conditions of artificial fertilization by momentary
warming and of normal fertilization by spermatozoa. ‘The fol-
lowing table gives the results of two series of experiments. Sper-
matozoa were added to successive portions of eggs, taken in each
series from a single female, at the indicated intervals after removal
from the animal. ‘The condition of the eggs at the time of fertili-
zation is indicated by the italicized portion in parentheses.

Four other similar series of experiments were performed with,
in general, very similar results. In all of these the most favorable
time for fertilization was either before or about at the time of the
separation of the first polar body; eggs fertilized at periods of one
to three hours after the completion of maturation gave few or no
larve, and these were mostly abnormal. ‘These experiments
agree in indicating that the egg gives the best response to the
fertilizing influence of the spermatozo6n at or near the time of
separation of the first polar body. After the separation of the
second polar body the proportion of developing eggs undergoes
rapid decline. It is however possible for eggs at such stages to
give normal larvz on fertilization, although the optimal conditions
are found at earlier stages.

On comparison with the results of momentary warming a cer-
tain agreement is seen. ‘The egg responds best to both fertilizing
influences at or near the time of separation of the first polar body
although rather before than after this event in the case of warming
This agreement is of some further interest as indicating that the
essential determining conditions of the initiation of the develop-

412 Ralph 8S. Lillie

mental process are similar in normal and in artificial fertilization.
Further and more precise analysis of these conditions is needed;
in particular, examination should be made of the susceptibility
of eggs, at different periods during and after maturation, to the

TABLE VI

Series I. August 24, 1907

I 35 m. | (First polar body not yet separated) Practically all mature eggs form
| blastule and gastrule

| 57 m. | (First polar body about to separate) A large proportion of larve;

| seems less favorable than Experiment 1

3 1h.17m. | (First polar body separated) Favorable; practically all eggs form
active larve

4 | 1h. 50m. (Mature eggs have both polar bodies) Somewhat less favorable than

Experiment 3; a large proportion of good larve

5 3h. 10m. (1 h. 15 m. after separation of 2d polar body) Marked contrast to
Experiment 4; most eggs dead and coagulated next morning; only
a few larve

Series II. September 2, 1907

Tr 50m. (First polar body not yet separated) Practically all mature eggs form

| larvae

(First polar body in all maturing eggs) Very uniform and normal
looking lot of larve; next morning are mostly active ; early gas-
trule

rh. 55m. (Both polar bodies in all maturing eggs) Less favorable than Experi-

| ment 2; larve less numerous and less well developed; a consider-

able number small or otherwise abnormal

4 2h. 55m. (More than one hour after separation of 2d polar body) Unfavorable;
| relatively few larve and these mostly abnormal; most eggs dead

| and coagulated next morning

| 4h. 30m. (Nearly 3 h. after 2d polar bodies) Still less favorable. Most

mature eggs are dead and coagulated next morning; a few larva,

mostly small, thick-walled, or irregular in shape. None normal

6 5h.25m. A few larve; most eggs dead and coagulated next morning

fertilizing influence of momentary warming in dilute potassium
cyanide solutions. This method, as will shortly be shown, pro-
* duces results far superior to those obtained by simple warming in
normal sea-water; and it is possible that after the completion of

Artificial Parthenogenests in Starfish Eggs 413

§S

maturation eggs may be found to respond to some such form of
treatment. As yet I have made no investigation of these relations.
Probably the most appropriate form of treatment will be found
to vary at different stages, according to the physiological condi-
tion of the egg. The experiments about to be described indicate
that the state of oxidation of the egg-protoplasm is a most impor-
tant factor; and it seems not unlikely that the above differences 1n
response at different periods may be found to depend largely on
varying conditions of oxidation at different stages.

Effects of Combining Momentary Elevation of Temperature with
the Action of Cyanide Solutions

The supposition that momentary elevation of temperature pro-
duces its effects on the eggs through an acceleration of oxidation
processes suggested itself early in anes investigation. ‘The beauti-
ful experiments of Loeb® had shown the importance of the presence
of oxygen in the action of hypertonic solutions on the Strongylo-
centrotus egg. I therefore tested the effects of warming starfish
eggs under conditions that exclude the influence of accelerated
oxidations. For this purpose sea-water containing potassium cya-
nide to +49 concentration was employed. In this medium intra-
cellular oxidations are greatly retarded if not almost altogether
suppressed, as shown by the fact that mature eggs remain for
days without undergoing the typical coagulative disintegration,
which, as Loeb has shown, is dependent on oxidations. In the
following experiments the eggs were warmed to 35° for 70 seconds
while in KCN sea-water, to which they were transferred in some
cases directly from normal sea-water, in others from 5%, KCN in
which they had been allowed to lie for varying periods of time.
After warming, the eggs were transferred in some experiments
directly to normal sea-water, in others to 3%), KCN at normal tem-
peratures, whence, after varying intervals, they were transferred
to sea-water.

The influence of previous treatment with cyanide solutions on
the development of eggs warmed momentarily in normal sea-water

® Loeb: Biochemische Zeitschrift, vol. i, pp. 189, 1906, ef seq., and preceding papers in University
of California Publications.

414 Ralph S. Lillie

was first tested. Sea-water containing KCN in 355 to pe Con-
centrations acts in the same manner as sea-water deprived of its
dissolved oxygen by a current of hydrogen or otherwise; the mat-
uration process is checked, and may be resumed on retransfer to
sea-water if too long aninterval has not elapsed. As shown above,
after the maturation-process has progressed beyond a certain
stage, starfish eggs become less and less susceptible to the influence
of momentary warming. It can be shown that the process (what-
ever its nature) which deprives the egg of this susceptibility is
retarded or prevented along with the maturation by the addition
of cyanide to the sea-water. ‘This is illustrated by the following
experiments :

Eggs were placed August 21, 1907, 20 to 25 minutes after
removal from the animal, in sea-water containing 3755 KCN. In
this solution they were left for 2h. 30 m. ‘They were then trans-
ferred to normal sea-water (which was changed to free the eggs of
adhering cyanide) and portions were warmed to 35° for 70 seconds
at successive intervals of ten minutes until the appearance of the
first polar body. At the close of the period of exposure to the
cyanide solution the eggs were almost all in an early maturation
stage with invisible germinal vesicle. Maturation was resumed
in normal sea-water; the polar bodies began to separate after an
interval of 1 h. 30 m.; a certain delay in the resumption of the
process is thus indicated. Eggs were warmed at the following
intervals after return from cyanide solution to normal sea-water
and the results were as tabulated in the following table:

TABLE VII
I 5m. | Eggs form membranes and some reach well-advanced cleavage
| stages. No larve formed
2 15 m. More favorable; a considerable number of larvae formed
3 | 25 m. Considerable number of active larve
4 35 m. Seems rather less favorable than Experiment 3; still a good propor-
tion form larve
5 45 m. Less favorable; only a few larve
6 thse isams | Unfavorable; no larve formed

1h.35 m. Unfavorable; no larve

Artificial Parthenogenests in Starfish Eggs 415

An experiment of August 17 showed a similar result: Eggs placed
ten minutes after removal in 37-5 KCN, left in this solution two
hours, then washed for 10 minutes in normal sea-water and
warmed, gave a considerable number of larve.

In these experiments the eggs were not warmed directly in the
cyanide solution; but were first transferred to normal sea-water
and then after an interval, subjected to the warming process in
the latter medium. The largest proportion of larvae developed
from eggs warmed within 15 minutes to half an hour after this trans-
fer (Experiments 2 to 4); later the conditions became less favorable.
The failure to reach advanced stages in Experiments 5 and 6 may
seem to contradict the rule found above that optimal conditions
for parthenogenesis are found at a time approaching that of the
separation of the first polar body. ‘The influence of the cyanide
must, however, be taken into account; as will be seen later the
presence of cyanide during the warming process improves the
conditions greatly, and the greater favorability in the earlier
experiments in all probability depends on the relative briefness
of the period succeeding removal fromthe cyanide solution. These
eggs were thus exposed at a relatively favorable stage of matura-
tion while still toa certain degree under the influence of the cyanide.
Sucha combination of circumstances would be favorable to devel-
opment.

In the experiments now to be described the eggs were exposed
to the high temperature while in the cyanide-containing sea-water.
In the first series they were placed in 559 KCN solution at an early
maturation stage, and after varying intervals were warmed to 35°
for 70 seconds in the same solution and then transferred directly to
sea-water. The result has appeared uniformly that under such
conditions a far larger proportion of eggs develop, and develop-
ment is more rapid and more nearly normal, than in eggs warmed
in normal sea-water without the cyanide treatment.

416 Ralph 8S. Lillve

The following series will illustrate:

TABLE VIII

August 24,1907. Eggs were removed at 2:55 p.m. and after 30 minutes in normal sea-water were transferred
to eat KCN in sea-water. In this solution, after the intervals indicated, successive portions were
warmed to 35° for 70 seconds, and immediately transferred to normal sea-water. In each experiment
the eggs were allowed to settle and the sea-water was changed and this washing process was repeated
a second time

Period in KCN
: RESULT
before warming
|
I 30m. | Almost all eggs form larvae, largely more or less irregular blastule;
| | some reach early Bipinnaria stage
2 | 50m. Larger proportion of active and normal larve than in Experiment

1; practically all mature eggs form larve of which many reach the
early Bipinnaria stage

3 th.1om. Rather less favorable than Experiment 2; many larve reach early
Bipinnaria
4 th.jom. Majority of mature eggs form larve a good many of which are small

and thick-walled; very active swimmers. A fair proportion reach
early Bipinnaria

5 2h.35m. Sharp contrast to Experiment 4; all eggs die in an early stage. No
larve formed

. . . 2 .
Control warmed in normal sea-water: Three portions were warmed in normal sea-water (without

previous cyanide treatment) at respectively 30, 40 and 50 minutes after removal. All three formed

numerous active larve; the conditions, however, were decidedly less favorable than with the cyanide-

treated eggs; most eggs died in early stages, development was slower, and the resulting larve were less
active and normal than in the favorable cyanide cultures.

Sperm-fertilized controls: Sperm was added to five successive portions at 35 m., 57 m., 1h. 17 m.,
1h. 50m. and 3 h. 10 m. after removal; numerous active ]arve were obtained in all but the last; on the
whole, the best sperm-culture was inferior to the best cyanide-culture and reached less advanced stages
of development.

A second series on August 27 gave similar results though the
eggs were not so favorable. The result was, however, all the
more striking since the best cyanide cultures were found to give
a larger proportion of active normal larve than were obtained
with sperm fertilization, even at the most favorable time.

Artificial Parthenogenests in Starfish Eggs 417

TABLE IX

August 27. Eggs were removed at 9:45 a.m.; left 30 minutes in normal sea-water; then transferred to
song KCN, and after the designated intervals warmed to 35° for 70 seconds in this solution, from
which they were transferred directly to sea-water ; this was changed twice to remove all traces of cyanide

Time in KCN |
solution beats
|
I 45 m. All maturing eggs form membranes and cleave to an advanced stage.
Only a few form blastule; these are relatively feeble and abnormal
2 60 m. | The majority of mature eggs form blastula; larve are largely abnor-
mal, with walls of unequal thickness; the number of active and
normal larva is greater than in the sperm-fertilized control
3 | rh.25 m. | Less favorable than Experiment 2. Eggs mostly stop short in early
cleavage stages; only a few larve obtained
4 1h. 45 m. | Still less favorable. Eggs cleave irregularly and very few form blas-
| tule
5 2h.15m. | Like Experiment 4; a few feeble abnormal blastulae
6 2h.45m. | Eggs stop short in early cleavage; no larve
7 3h. 50m. | Like Experiment 6. Cleavage irregular; no larve

Control warmed in normal sea-water: Three portions warmed respectively 30, 40 and 50 minutes after
removal gave only a few small abnormal blastule.

Sperm-fertilized control: Portions were fertilized 30 m., 49 m., I h. 11 m., 1h. 27 m., rh. 55 m., 2
h. 55 min., 4 h. 20 m. after removal; in the best cultures (30 m. and 49 m.) only one-third to one-
half of the eggs formed blastule of which a large proportion were abnormal.

In experiments 3 to 7 many eggs cleave irregularly and stop short in early cleavage stages. A remark-
able peculiarity of such eggs is that after 24 hours the blastomeres still remain clear and uncoagulated
and apparently living, though undergoing no further cleavage. This condition is in striking con-
trast to the fate of eggs fertilized either normally or artificially without cyanide treatment and whose
development also ceases in early stages; in such eggs the blastomeres rapidly undergo the typical coagu-
lative disintegration characteristic also of mature unfertilized eggs. The cyanide has apparently per-
manently modified the cell-protoplasm in such a manner as to check or prevent the oxidations on which

this breakdown depends.

A third similar series (August 23) should also be mentioned
briefly. In this series the control eggs, warmed in normal sea-
water without previous cyanide treatment, gave no swimming
larve; and the sperm-fertilized eggs gave only a few, from a por-
tion fertilized about 40 minutes after removal; these fertilized
later (Ih. 10 m., 1 h. 25 m., rh. 50m, 3 h. 15 m.and 5 h. 15 m.)
gave no larve. The eggs were thus typically “unfavorable.”
A portion of the unfertilized eggs was placed in 3;%;5 KCN 30 min-

418 Ralph §. Lillie

utes after removal, warmed to 35° for 70 seconds after the indicated
intervals in the cyanide sea-water, and then transferred as above
to fresh sea-water. ‘The results were as follows:

25 m. in KCN | Large proportion of vigorous larve formed

z 55 m More favorable than Experiment 1; after 24 hours numerous blastule
and gastrule were present

1 h. 30 m. | Less favorable; relatively few blastule were formed and these were
3 3 | y
| mostly abnormal
|
|

4 2 h. 50 m. Unfavorable; very few eggs reach blastula stage

A fourth series (August 29) gave an even more striking result.
Eggs were placed, 40 minutes after removal, in >; KCN, warmed
to 35° for 70 seconds after the following intervals in this solution,
then transferred to normal sea-water which was changed as usual.
The control eggs warmed in sea-water at 40, 50 and 60 minutes
after removal gave only a few blastula, the great majority dying
and disintegrating at an early stage. In the best of the several
sperm-fertilized portions only one-third to one-half of the mature
eggs formed blastulae which were largely feeble or otherwise
abnormal. ‘The results were as follows:

I 35 m.in KCN | Next morning the dish was full of vigorous normal-looking blastule
and early gastrule; condition much better than in the best sperm-
fertilized control

2 60 m. Decidedly less favorable than Experiment 1; a good pro portion of
eggs form larvee, but these are less active and normal than above
3 1h. 35 m. Still less favorable; nevertheless a large proportion have formed
larve; these are largely irregular in form and somewhat feeble

in movement

In each of the above four series of experiments a far larger
proportion of eggs produced larvae after treatment with cyanide
for an appropriate length of time than after simple warming un-
accompanied by such treatment; and the development was more
nearly normal and resulted in the production of larger and more
vigorous larve. ‘[heresults were indeed comparable in the best in-
stances to those obtained with normal sperm-fertilization; in fact, in
the last twoseries better conditions were obtained with the artifici-
ally fertilized eggs than with those fertilized in the natural manner.

Artificial Parthenogenesis in Starfish Eggs 419

It is noteworthy that a certain time of exposure to the cyanide
solution—apparently about one hour or somewhat less—produces
optimal conditions for development; after more prolonged expo-
sure warming tends to result in abnormal development; in Tables
VIII and IX the proportion of eggs that reach a larval stage is seen
steadily to diminish with increase in the time of exposure to the
cyanide beyond an hour or so, and the larve tend to become
thick walled, irregular in shape, or otherwise abnormal. After
exposure for more than two hours to the cyanide few eggs develop
to a free-swimming stage. ‘This change in the condition of the
eggs points to the existence of certain processes other than oxida-
tions which continue unchecked in the presence of cyanide;
there are no doubt hydrolyses of various kinds, and it may reason-
ably be inferred that both kinds of processes are concerned in the
changes that render the egg capable of parthenogenetic development.
Suppression of oxidations for a time, during which the hydrolyses
proceed unchecked, appears then to be favorable to bringing the
eggs into a condition in which they respond readily to momentary
warming; but if the hydrolyses unaccompanied by oxidations are
allowed to proceed too far, lack of codrdination in the succeeding
developmental processes seems to result, as shown by the increased
proportion of unfavorably developing eggs. Normally a certain
balance between the oxidative and the hydrolytic processes must
exist; possibly a disturbance of this balance may be an important
condition in the initiation of the developmental process. Such
an interpretaton is at least suggested by the foregoing results.

It should be pointed out that simple exposure to cyanide solu-
tions without warming has no influence in initiating development
in these eggs—at least under the above conditions. In the second
of the two series tabulated above a portion of eggs was transferred
from the cyanide solution to sea-water, without warming, at the
time of each experiment of the series. None of these eggs formed
membranes or showed any other sign of development and all were
dead and coagulated next morning. The momentary elevation
of temperature is thus essential. Since hydrolytic processes are
relatively unaffected by cyanide, we may infer that hydrolyses are
accelerated to at least four or five times the original velocity during

420 Ralph §. Lillie

the period of warming—probably to an even greater degree, since
the above results on membrane-formation (pp. 381, et seq.) indi-
cate a much higher temperature-coefhcient of acceleration for such
processes under the conditions prevailing inthe cell. Indications,
then, seem to point to an acceleration of hydrolytic processes,
combined with a repression of oxidations, as an important con-
dition in the initiation of development in these eggs. That hy-
drolyses are in fact accelerated seem to be here ii the condi-
tions of the membrane-formation; this event occurs quite normally
in the cyanide solution; it appears to be dependent on a hydrolysis
which is greatly accelerated by a rise of temperature; and presum-
ably other hydrolyses in the egg would be similarly affected by
the same change of conditions. Membrane-formation seems to
afford a clear proof that certain processes, not dependent on oxida-
tions, are markedly accelerated by momentary warming, and that
certain critical changes in the developmental capabilities of the
egg- protoplasm may result from such momentary acceleration.

Naturally it is impossible for eggs treated as above to develop
while remaining in the cyanide solution; the transfer to oxygenated
sea-water is indispensable. ‘This transfer however need not be im-
mediate. It is possible to keep eggs, after warming under the above
conditions, in cyanide sea-water or a certain not too prolonged
period before transfer to sea-water. No visible change occurs
during the stay in the cyanide solution, but on transfer to normal
sea-water development proceeds normally. Indeed, under cer-
tain conditions such after-treatment with cyanide has proved
highly favorable to development as the following experiments
illustrate :

In these experiments the eggs, after remaining for a certain
time in cyanide-containing sea-water, were warmed momentarily
as above and brought to normal temperature in that medium; then
after an interval they were returned to normal sea-water. A cer-
tain stay in the cyanide solution after warming proved in every
case decidedly favorable.

Artificial Parthenogenesis in Starfish Eggs 421

The following series will illustrate :

TABLE X

Series I. September 7. Eggs were transferred 30 minutes after removal from the animal, to sea-water
containing ,M.. KCN; after an interval of ca. 40 minutes they were warmed in this solution to
35° for 70 seconds; thence transferred to cyanide solution at normal temperature; from this, after the

designated intervals, portions were transferred to normal sea-water.

Exposure to KCN after
Spent RESULT
1 | (control) 0 (to sea-water | Not favorable; comparatively few larve formed
directly )
2 5m. A striking contrast to the control; nearly all mature eggs form active
larve; the majority of these gastrulate and many reach the early
Bipinnaria stage
3 1om. Similar to Experiment 2; very favorable; numerous early Bipinnarie
result (with mesenchyme and with the three intestinal divisions
plainly marked)
4 20 m. A very good vigorous lot of gastrula were obtained, but rather less
favorable than in Experiments 2 and 3; relatively few reach ad-
vanced stages

Controls warmed in normal sea-water 35, 45 and 55 minutes after removal gave considerable numbers
of good larve.

Sperm-fertilized control, fertilized one hour after removal, gave also a large proportion of larve, though
fewer than in the best experimental cultures; development was also less rapid.

A number of eggs were left in the KCN solution after warming until next morning (23 hours); they
were then clear and uncleaved and all had typical membranes. On transfer to normal sea-water none
underwent development, and next day all were dead and disintegrated.

In the above series of experiments a marked increase in favor-
ability resulted from the brief after-treatment with cyanide. In
those next to be described a greater range of exposure to the cya-
nide solution was employed; otherwise the procedure was the
same.

422 Ralph S. Lillie

TABLE XI

September 2,1907. The eggs were left in sea-water for 45 minutes after removal; then transferred to .M >
KCN for one hour, warmed to 35° for 70 seconds in this solution, retransferred to ,M., KCN at
normal temperature, and thence, after the designated intervals, transferred to normal sea-water which

was changed twice to remove all cyanide.

| i]
[Interval between warm-
ing and return to Condition after 24 hours

pormal sea-water

1 | o(control; directly to sea-| Large: number of normal well-advanced gastrule

water after warming)

2 4m. A decidedly larger proportion of swimming larve than in Experiment
1; numerous normal gastrule are formed

3 14m. | A large proportion of larva; on the whole less uniform and less
advanced than in Experiment 2

4 24m. | Like Experiment 3 but with somewhat larger proportion of abnor-
malities; still, many good active gastrule

5 44m. Distinctly less favorable than Experiment 4; fewer larve than in
Experiments 3 and 4, mostly blastule or imperfect gastrule

6 64m. Similar to Experiment 5; a good proportion of larva, largely abnor-
mal; fair number of gastrule

7 1hr.24m. Relatively unfavorable; a smaller proportion of larve and these

mostly small thick-walled blastule; relatively few gastrula, which
are less advanced than in above experiments

8 2h. 34m. Considerable number of thick-walled blastule, but fewer than in Ex-
periment 7. No regular blastula and no gastrule. Many eggs
have stopped short in early cleavage stages

9 23 h. Development stops in early stages and eggs disintegrate; none reach

larval stages

Controls warmed in normal sea-water, 50 and 65 minutes respectively after removal, gave a fair num-
ber of blastule after 24 hours, of which a few were beginning to gastrulate. As compared with Experi-
ments 1 to 6 above, the larve are fewer and in a less advanced stage of development.

Of the sperm-fertilized controls, those warmed within 1 h. 30m. after removal gave a large number
of normal active larve.

On examination, after 24 hours, of eggs left in the cyanide solution, all were found with membranes,
round, clear, uncoagulated and uncleaved; many, however, showed little pseudopodia-like projections,
and frequently small portions of the surface-protoplasm had become detached from the egg. While
cleavage is impossible in the KCN solution, there appears nevertheless to have been some slight cyto-
plasmic activity in these eggs.

In a third series similar conditions were found; in this series
the eggs were unfavorable and very few larve resulted even in the
best sperm-fertilized control. A relatively small proportion of eggs
formed larve in the best experiments; still, exposure to 53,5 KCN

Artificial Parthenogenests in Starfish Eggs 423
solution for some minutes after warming gave decidedly better re-
sults than were obtained from eggs transferred directly to sea-water
without after-treatment with cyanide. The eggs were removed
from the animal at 10 a.m. September 4, 1907; at 10:35 they were
placed in 5; KCN; and after 55 minutes were warmed to 35°
for 70 seconds and then replaced in cyanide solution at normal
temperature, whence, after the intervals used, they were transferred
to normal sea-water. Here the eggs brought into sea-water
directly after warming in cyanide solution gave no larvae; while
eggs after exposed to cyanide for only 5 minutes yielded consider-
able numbers of good gastrulz, proving in fact more favorable
than the best sperm-fertilized control; 10 minutes after-treatment
on the other hand gave few larvae; and eggs left respectively 20,
35 and 50 minutes in cyanide after warming gave successively
fewer and fewer; while none resulted with after-exposures of
th. 10m., rh. 30 m., 2h. 50 m. and 4h. 20 m.

These experiments indicate clearly that checking of oxidation
processes during a certain interval after warming acts favorably
under certain conditions; if this interval is prolonged for more

han a few minutes conditions become rapidly less favorable,
possibly, as suggested above, in consequence of the progress of
certain hydrolytic processes unaccompanied by oxidations. ‘The
striking increase in the proportion of developing eggs under the
treatment used above, and also in the rate and normality of the
development, suggests strongly that anaérobic conditions—at
least at certain stages—form an important factor in the initiation
of development in starfish eggs. Oxygen is necessary to the
developmental process itself; but the internal changes that impart
to the egg the distinctive power of automatic development seem
best induced under conditions that must very effectually prevent
most intracellular oxidations—at least those conditioned by the
presence of enzymes. ‘The above results indicate therefore that
momentary elevation of temperature—assuming that its essential
action 1s the acceleration of chemical processes in the egg-sub-
stance—must affect primarily other processes than the oxidative;
in brief, acceleration of these processes, presumably hydrolytic in
nature, simultaneously with a suppression of oxidations, appears

424 Ralph S. Lillie

in some manner to result in changes leading to the initiation of
development.

After-treatment with cyanide also acts favorably in the case of
eggs that have been warmed in normal sea-water without previous
exposure to cyanide solutions. The following experiments will
illustrate :

TABLE XII

September 9, 1907. Eggs were removed at 10:15 a.m. and the majority began to mature. After 43
minutes they were warmed in normal sea-water to 35° for 70 seconds. One portion (A) was then
transferred to normal sea-water; a second portion (B) to 5 — KCN solution, and from this portion

were transferred at the following intervals to sea-water

eee ye
Interval in KCN solution
|

before transfer to RESULT

sea-water
I © (control A; untreated) Almost all eggs die in early cleavage stages; only one or two blastule
with KCN) | found
2 5m. | Most eggs die, but a distinctly larger proportion reach the blastula

10 m. | Similar to Experiment 2; larve are decidedly more active, numerous,

| | stage than in the control and these are better developed

and well-developed than in the control; some have entered the
early gastrula stage after 24 hours
4 | 25m. | Conditions are still more favorable; larva are more numerous and
| more typical than in Experiments 2 and 3
5 60 m. Similar to 4; good many early gastrule (mostly more or less abnor-

mal) after 24 hours

A portion of eggs fertilized with spermatozoa about one hour after removal gave a good proportion of
larve; largely small and thick-walled or otherwise abnormal. The eggs were thus not especially favor-
able.

The proportion of eggs developing to blastule and farther,
while not large in the above series, was decidedly increased by the
after-treatment with cyanide, and development proved both more
rapid and more nearly normal in eggs thus treated. The best
conditions were found in Experiments 4 and 5. Too prolonged
after-exposure to cyanide affects the egg injuriously, the propor-
tion of abnormal larve being greater in Experiment 5 than in
Experiment 4.

A repetition of this experiment, with a larger range of exposure
to cyanide, gave a similar result (Table XIII).

Artificial Parthenogenesis in Starfish Eggs 425

TABLE XIII

September 10, 1907. Eggs were removed at 10:30 a.m.; the majority underwent maturation. After 45
minutes the eggs were warmed to 35° for 70 seconds as usual; a portion (for control) was placed

immediately in normal sea-water; the remainder in KCN solution, whence, after the intervals

M
Zo000

indicated, portions were tran ae 10 sea-water

Time in KCN solution | RESULT

™

0 (control) Nearly all eggs are dead after 24 h., but a few blastule and gastrule
| have developed. (A second portion of eggs warmed about 65 m.
after removal also gave a few larve, mostly irregular blastule)

2 6 m. Most eggs die but larve are distinctly more numerous and active than
in the control; a fair proportion are gastrulating after 24 hours.

3 | 11m. | Similar to Experiment 2; a large proportion of larve are gastrulating
after 24 hours.

4 21m. Rather less favorable than Experiments 2 and 3; a fair number of

larve formed

5 36 m. Similar to Experiments 2 and 3; a fair proportion of larve are gas-
trulating after 24 hours

6 | 59m. A good proportion of larve after 24 hours, largely well formed

| early gastrule
ap || 1h. 30m. Unfavorable; no larve found
8 4h. Unfavorable; no larve

The Sperm-fertilized control (sperm added 40 m. after removal) gave a large proportion of gastrule
largely abnormal—irregularly shaped, thick-walled, or sluggish. Very few have gastrulated by 24
hours.

Here also a decided increase in favorability followed after-
exposure to the cyanide solution for a not too prolonged period.
The results however were less favorable than in the experiments
where eggs were exposed to cyanide for some time previously to
warming eid were warmed in the solution. We may infer that
while suppression of oxidations for a certain period after warming
is favorable to development in eggs which have previously been
well exposed to oxygen, this treatment differs from the preceding
in certain very essential particulars, the nature of which requires
further analysis. ‘Treatment with cyanide previously as well as
subsequently to the momentary warming is essential if the most
favorable conditions are to be attained.

On reviewing the general outcome of the experiments described
in this section we are led, first, to the conclusion that the entire
series of events leading to the initiation of development in these
eggs includes the changes preceding and following the warming

426 Ralph S. Lillie

process, as well as those immediately induced by the latter.
Secondly, all of these changes appear to proceed best under con-
ditions of lack of oxygen—in other words, to be essentially anaé-
robic in their nature. A predominance of anaérobic processes in
the changes initiating development implies that an important
part is played here by reductions (in the chemical sense), since
anaérobic metabolism is always accompanied by the production
of strongly reducing substances. ‘The possible part played by
such reductions in the processes of cell-division and growth has
been discussed by Mathews in the paper already cited; and the
above general result is therefore consistent with his view that the
production of asters (regarding this phenomenon as an essential
feature of mitosis) is the expression of localized reducing processes.
I can however hardly see my way clear to the conclusion that the
momentary elevation of temperature under anaérobic conditions
acts essentially by accelerating reductions and thus producing
astral areas. Whilethis isa possible interpretation, it can, asalmost
purely speculative, serve no particular purpose at present until
confirmed or disproved by experiment. Moreover, in the sea-
urchin egg the conditions seem of quite an opposite nature. Still,
so far as regards the main chemical conditions of the partheno-
genetic initiation of development in the starfish egg, the above
results appear to indicate very definitely a subordination of oxida-
tive processes to those of some other nature.

This conclusion, while opposed to that reached by Loeb in the
case of the sea-urchin egg, is in harmony with the recent experi-
mental results of Delage® with the starfish. In this form partheno-
genetic development through the action of carbon dioxide was
found to be best obtained in thie absence of oxygen; a high concen-
tration of oxygen in the carbon-dioxide-containing sea-water
proved definitely unfavorable; and, in general, the lower the pro-
portion of oxygen present, the better were the results obtained.
Thus the initiation of development through this means, as well as
through momentary warming, appears dependent on processes
of an essentially anaérobic nature. Precisely contrary relations

33 Delage: Comptes rendus, vol. 145, p. 218, 1907.

Artificial Parthenogenesis in Starfish Eggs 427

were found by Delage in the case of Strongylocentrotus, as had
already been determined by Loeb; here the presence of oxygen in
the hypertonic solutions is favorable to development. We have
thus a striking contrast between the two forms in respect to the
part played by oxygen in the initiatory process. ‘This contrast
cannot be explained at present; it can only be referred to deep-
seated constitutional differences between the two eggs. One fur-
ther consideration is suggested and should be emphasized here:
it must be recognized clearly that the physiological conditions
underlying the initiation of development—i. e., the bringing of the
egg into a condition in which it becomes capable of automatically
passing through its characteristic ontogenetic cycle—may be of
quite different nature from those on which the developmental proc-
ess itself depends. ‘This is seen in the fact that notwithstanding
the contrast in the conditions of the initiatory process, both the
above eggs require the presence of free oxygen for their develop-
ment. Unexplained constitutional differences between species
play a part here, and we are not yet in a position for broad general-
ization. Nothing but further exact investigation of the conditions
of artificial parthenogenesis in eggs of different groups can be
expected to bring to light the fundamental conditions common to
the different types. For the solution of this problem a system-
atically inductive ._procedure seems safest at present.

SUMMARY

1 Momentary exposure of the eggs of Asterias forbesi, dur-
ing the early maturation period, to temperatures of 35° to 38°
results in the formation of typical fertilization membranes, fol-
lowed by the development of many eggs to a free swimming
larval stage.

2 The favorable duration of exposure to the above tempera-
tures is very brief, with a well-defined optimum for each tempera-
ture; this optimum is approximately 70 seconds for 35°, 40 to 50
seconds for 36°, 30 seconds for 37°, and 20 seconds for 38°. A
very rapid rate of decrease in time of exposure with rise in tempera-
ture is thus indicated, a rise of three degrees above 35° apparently

428 Ralph 8S. Lillie

tripling the velocity of the process or combination of processes on
which the initiation of development depends. The process of
membrane-formation shows a similarly high temperature-coefh-
cient of acceleration.

3 The responsiveness of eggs to this treatment varies greatly
at different periods in the life of the egg. Warming within five
minutes after the removal of the eggs from the animal is ineffective,
and has the effect of preventing permanently the dissolution of the
germinal vesicle. Warming at any time between the beginning
of nuclear dissolution and the separation of the first polar body
may result in development and the production of larva; the most
favorable period is some little time (10 to 20 minutes) before the
separation of the first polar body. Warming subsequently to this
event tends to produce abnormal form changes or irregular cleav-
age; after maturation is complete the effect is mainly to acceler-
ate the coagulative change characteristic of mature unfertilized
eggs in presence of oxygen.

4. Maturing eggs placed in 5, KCN solution retain for several
hours their susceptibility to development by the above means. A
stay of a certain duration in cyanide solution followed by momen-
tary warming in this solution and transfer tosea-wateris followed
by a striking increase in the proportion of favorably developing
eggs. Further exposure of eggs to cyanide solution for a certain
period after warming effects a still further improvement in the con-
ditions of parthenogenetic development. Eggs thus treated with
cyanide approximate closely, in the rate, character, and favorability
oftheir development, to normally fertilized eggs.

5 Since the essential action of the above dilute cyanide solutions
is to prevent intracellular oxidations, the inference is drawn that
anaérobic processes play an important part in the series of changes
leading to the initiation of development in starfish eggs. Sup-
pression of oxidative combined with acceleration of hydrolytic and
reducing processes is indicated as a condition of the initiatory
process in these eggs.

THE SEX RATIO AND COCOONING HABITS OF AN
ARANEAD AND THE GENESIS OF SEX RATIOS?!

BY
THOS. H. MONTGOMERY, Jr.

Witn Two Ficures

This communication presents a study of the numerical propor-
tions of the sexes in Latrodectus determined for 41,749 newly
hatched young, with briefer observations on such proportions in
other spiders; then an account of the general cocooning habits;
next an attempt to show that different species of organisms prob-
ably have different sex ratios, with an explanation of the origin
of such differences.

I LATRODECTUS MACTANS FABR.

This is the largest North American Theridiid and it was selected
partly because of the ease with which it may be kept, but more
particularly on account of the great degree of sexual dimorphism:
with the adult males and females so different in form and size
it was anticipated that the sexes might be distinguished at the time
of hatching, and this hope was realized.

At Austin, Texas, where I have been observing these spiders,
the web of this ‘species is found usually on the ground beneath
a stone or log, sometimes several feet up within a crevice of a rock
wall. The female remains at the upper portion of the web, and
uses a niche or cranny as a retreat. Her web is composed of
unusually powerful threads, capable of holding the strongest
beetles and even of sustaining small stones; indeed I allowed my
captives to fasten down with it the glass plates serving as covers
for their cages, and this they did so firmly that the glass would
not fall when the cages were inverted. Adult males I have found

‘ Contributions from the Zodlogical Laboratory of the University of Texas. No. 89.

Tue JourNat or ExperiMENTAL ZOOLOGY, VOL. V, NO. 3

430 T hos. /5b Montgomery, tie

only on the webs of females and only from December to February.
Here, accordingly, the beginning of the reproductive season is in
the early portion of the year. But on the Colorado river about
sixty miles northwest of Austin I collected adult males in August.
It would seem then that different groups of individuals show dif-
ferent mating periods.

Methods

It was my object to determine not only the general sex ratio of
this species, but also the ratio for each successive cocoon of a given
spider. Therefore it was necessary to keep females through a
whole reproductive season. That the captivity of the mothers
did not produce abnormal results will be shown later. Only by
controlling individuals in this way can one obtain accurate notes
of the times of making and hatching of each cocoon, and also pre-
vent the cocoons from being parasitized. Early in March and
April of this year (1907) I collected a number of females and these
I have kept until the autumn, up to the close of the time of ovi-
position. ‘To each of them was devoted a separate cage of paste-
board, most of these cages about three inches high, and most of
them triangular with each side about three inches long. A photo-
graphic glass plate was used as a cover, and another asa base. All
these cages were kept together in a portion of my study where no
direct sunlight reached them, and upon the cover of each was laid
a paper card that excluded most of the light entering from above;
by lifting this card one could look into the web without injuring
it or disturbing the inmate. The bodies of their victims, when
they have sucked them dry, the spiders drop out of the web; fort-
nightly, accordingly, I pulled out the bottom glass plates so as to
remove these accumulations. Cocoons were also taken out from
below, by removing the same plates. “Thus the upper portion of
the snare where the spider awaits her prey and where she devours
It, was never disturbed, and to spare this web as much as possible,
food was admitted through a small hole in one side of the cage,
this hole being otherwise closed by a cork.

Only living food is accepted, and for this I used large house
flies caught in the usual wire traps; sometimes the diet was varied

Sex Ratio of an Aranead 431

by larger insects. ‘The spiders were given equal amounts of food,
and from the beginning of the experiment until August 8 all were
richly fed, and daily except in the colder portion of the spring
when food was hard to obtain, so that each of them averaged
probably five or six blue bottle flies a day, quite the equivalent
of the amount in a state of nature. Between August 8 and 29
they received only two meals, for I was absent; and in September
they received only three good meals. Up through the first week
in August, which marked the close of reproduction with most of
them, these spiders were kept under natural conditions of light,
temperature and amount of food. The healthy and active con-
dition of the captives until the middle of August, and the large
number of cocoons they produced, evidenced the favorable circum-
stances under which they were maintained.

Each spider received a separate number, and each cocoon the
number of the mother together with the cocoon letter; thus the
first cocoon of spider 2000 was 2000A, the second, 2000B, and so
on. No cocoon was removed from a cage until several hours after
its construction, for when just made the danger of injury to the
eggs is greatest; each was lifted out as gently as possible with a
pair of forceps, placed in a bottle covered with perforated paper
and kept there until the young emerged; these were preserved in
80 per cent alcohol within twenty-four hours of hatching. It is
necessary to kill the spiderlings before their first postnatal moult,
else they commence to attack each other. ‘This isolation of the
cocoons is the only method for preventing the young from dis-
persing and so becoming lost at the time of hatching.

Three series of females were kept: (1) twelve individuals whose
young were allowed to hatch for the computation of the sex ratio;
(2) five individuals whose eggs were preserved twenty-four hours
after oviposition to test possible voluminal differences; and (3)
two individuals kept to test parthenogenesis. More than this
number I could not keep well fed. No deaths occurred until
August 29, and the nine deaths from then on were probably due to
insufhicient feeding commencing with the second week of August.

432 Thos. H. Montgomery, fr.

Cocooning Habits

The mating I have not observed, but it probably takes place
about the beginning of the year when the adult males are found
upon the webs of the females. Wild cocoons are to be discovered
as early as February. The cocooning season extends, at Austin,
from that month continuously into August. I conclude that it
usually terminates in August, for only eight cocoons were made
by my spiders after the eighth of that month; of these two were
made in September and one in October. Further the last cocoons
of a series, especially such dating from the middle of August, are
frequently infertile; compare on Table I the last cocoons of 2013,
2016, 2021 and 2031. Females live on after the cocooning season
provided they are well nourished.

The completed cocoon is not quite globular but somewhat pyri-
form, the upper portion having a short stalk to attach it to the
object that overarches the web; when fresh it is snow white, when
older, yellowish, and its outer coat is markedly resistant and firm.
The process of cocooning and oviposition has much resemblance
to that of Theridium? but Latrodectus is less specialized in that
she applies the thread mainly by direct application of the spinner-
ets and rarely by manipulation of the fourth leg pair. The case
as seen in the making of cocoon 2020E was as follows: At 5:37
p-m. the mother was seen working at the base, a disc of flossy silk
then only 2 mm. in diameter; she hung below it, holding its edges
with her three posterior pairs of legs while with her first pair she
suspended herself from the web; she was then making 52 appli-
cations of her spinnerets per minute. The base was completed
at 6:04, an inverted cup with a diameter equal to that of the fin-
ished cocoon. Oviposition, with rapidly repeated uplifts of the
abdomen against the concave surface of the base, lasted from
6:04 to 6:16. The construction of the cover of the cocoon occu-
pied from 6:16 to 8:19; for the first ten minutes the fourth pair
of legs were used to comb out the thread before each application
of the spinnerets to the cocoon, after that time these legs were no

? Compare Montgomery: The oviposition, cocooning and hatching of an Aranead, Theridium tepi-
dariorum C. Koch. Biol. Bull., xii, 1906.

Sex Ratio of an Aranead 433

more employed to handle the issuing thread. The rapidity of
the applications of the spinnerets was found to be as follows:

from 6:16 to 6:25, 78 applications per minute,
from 6:25 to 6:45, 120 applications per minute,
from 6:45 to 7:30, 125 applications per minute,
from 7:30 to 8:19, 140 applications per minute.

At 8:19 she ceased suddenly, perhaps from exhaustion, then spun
again at the rate of 108 applications per minute from 8:28 to 8:32.
The cocoon was then completed, and the final touches were to
anchor it firmly in the web after cementing it to the roof. Now
each time the spider applies her spinnerets to the cocoon she draws
out a thread having a length of 5 mm. (the length of the fourth
tibia); multiplying the distance of such a thread by the number of
applications of the spinnerets, the astounding fact is reached that
in spinning the cover alone of the cocoon the spider employs a
thread having a total length of about eighty meters. “The mus-
cular energy employed is very great, being a rapidly repeated
uplifting of the heavy abdomen. Another spider worked on the
cover of a cocoon for one hour and fifty minutes, and two others
for five hours each.

Oviposition usually takes place in the morning before 6:30
o'clock, and a little later than that one usually finds the process of
cover making. In 143 cases oviposition was between midnight
and 7 a.m., in eleven cases between 7 a.m. and noon, in eight
cases between noon and 6 p.m., and in only one case between 6
p-m. and midnight.

The young make their own way out of the cocoon, usually
through a single circular aperture that they make probably by
biting; they emerge in rapid succession, and unlike the adults
are positively phototropic. In eight cases the hour of emergence
was between midnight and 6 a.m., in twenty-eight cases between
6 a.m. and noon, in sixty cases between noon and 6 p.m., and in
thirty cases between 6 p.m. and midnight. ‘The afternoon at its
hottest hours, between 3 and 5 of the clock, is the most frequent
time of hatching. The young do not commence cannabalism
until after their first postnatal moult, and the time of this varies

434 Thos. H. Montgomery, ‘fr.

with the temperature as well as with the individual spiderling.
Most of those cocoons laid from the middle of June on hatched
in nineteen or twenty days, and two in as short a time as seventeen
days; longer intervals are characteristic of eggs laid earlier in the
year, and the earliest cocoon always takes the longest time to
hatch; this is readily seen on comparing successive cocoons in
Table I, and shows that the rate of development depends directly
upon the temperature.

The total number of cocoons raised by those seventeen spiders
that furnished series of them was 187, an average of eleven to each
spider. One individual formed eight cocoons, one formed nine,
four formed ten each, four formed eleven each, five formed twelve
each, while two formed thirteen each, the range thus extending
from eight to thirteen. The time interval between successive
cocoons varies with the month, so probably with the amount of
nourishment, it being shortest in July and August; such intervals

may be easily compared from the data given in Table I.

Sexual Dimorphism and the Sex Ratio

On the North American continent there are two good species
of the genus Latrodectus Walck., L. mactans Fabr. and L. geo-
metricus Keys., as I have convinced myself by a study of the
material in the United States National Museum; for the oppor-
tunity of examining this collection my thanks are due to the cour-
tesy of Mr. Nathan Banks. ‘This collection contains specimens
of mactans from California (San Bernardino, Tulare county,
Clemente Island), Texas, New Mexico, Nebraska, District of
Columbia, Colorado, Georgia, North Carolina and Oregon;
while Marx’ states that it occurs also in Pennsylvania, Ohio and
Utah. Though mactans shows this wide distribution and is every-
where of rather confined local occurrence it does not appear to
have split into geographical races.

The sexes of mactans show a marked dimorphism both in size
and color, as seen in the following comparison.

3 Catalogue of the described Aranee of temperate North America. Proc. U.S. Nat. Mus., xii, 1890

Sex Ratio of an Aranead 435

Adult female. Maximum dimensions: abdomen from anterior
convexity to spinnerets, 12 mm.; first leg, 21 mm., second leg, 15
mm.; third leg, 13 mm.; fourth leg, 20 mm. Rufous black is the
color of the cephalothorax, sternum and legs, and only the meta-
tarsi and tarsi are lighter. “The abdomen is deep black, enormous,
nearly globular and arched on all surfaces except the ventral; it is
marked only by a broad red mark on the venter behind the epi-
gynum, a short red median band just dorsal to the spinnerets,
and (rarely) traces of other red spots along the dorsum; at the
dorso-anterior border are one or two narrow transverse red marks.
In a few specimens from more northern localities the dorsal red
spots were prominent, and in one only a pair of oblique red bands
on each side. The female is thus shining black with a few red
markings.

Fia. 1

Adult male. As shown by the accompanying figure the male is
much smaller than the female, with elongate abdomen; in this
figure the abdomina are shown from ventral and lateral views and
the stippled areas denote the light markings; he has also propor-
tionately longer and more slender legs. “The cephalothorax and
sternum are both pale brown with darker borders, the cephalo-
thorax also with a darker median stripe. The legs are yellow;
the distal ends of the femora and patella are darker, and there are
two rings of the same dark color at the distal ends of the tibiz.
The abdomen in seven specimens has a broad white dorso-median

436 Thos. H. Montgomery, fr.

band extending from near the anterior end to the spinnerets; at the
antero-dorsal boundary a transverse white band that extends
down on both sides; on each side behind the latter are two oblique
white bands; all these bands are narrowly edged with black. On
the venter there is a broad white mark which ts on each side bor-
dered by black, and on each side of the spinnerets are three oblong
black spots. The remainder of the abdomen is pale brownish
flecked with white. In one male there was a red line inclosed
within the dorso-median white band of the abdomen, and deep
black filling all the spaces between the abdominal white bands.‘

The males in the instar just preceding the adult stage have
the abdomen larger and more rounded, the legs proportionately
thicker, and the abdomen colored like the young female, namely,
dull or deep black, with a medio-dorsal white band including a red
one, an arched transverse white band anteriorly, two oblique
narrow white bands on the sides, and a broad white band on the
venter.

The newly hatched of both sexes are yellow with black stripes
on the abdomen as follows: two narrow parallel stripes along the
dorsum, two broader ones on the venter, and three (often broken)
oblique stripes on each side. Thus the color of the adult male
retains the color pattern of the young much more than does the
adult female, for the latter becomes to great extent deep black.
In color, size and activities the male is decidedly more embryonic.

So far all observers, with the exception of Doumerc, have held
the newly hatched of spiders to be sexually indistinguishable.
At that period the genital plates are quite simple, and the pedipal-
pal tarsus of the male is not different from that of the female. On
sectioning the spiderlings of L. mactans I could not distinguish

4J have described mactans rather more fully than might seem necessary for our present purposes, but
this is called for on account of the present confusion with regard to the American species. For this
reason I will give briefly the characters in which geometricus differs from mactans. The male of geo-
metricus differs only in having two pairs of black spots just behind the middle of the dorsum. The
female of geometricus differs from the female of mactans in having the dimensions of all parts of the
body slightly smaller but the abdomen much smaller, the legs pale colored with dark rings; in geometricus
also the general color of the abdomen is pale brown, more rarely black, and always marked by lighter
markings on the dorsum and sides. Further, the cocoon of geometricus has the surface beset with

numerous slender, cylindrical villi, each from 1 mm. to 1.5 mm. in height, while the cocoon of mactans
is quite smooth. L. geometricus occurs in California and Jamaica.

Sex Ratio of an Aranead 437

ovaries from testes, for each is simply a small paired chord of
germ cells of an early generation. But careful comparisons dem-
onstrate that there are two constant forms of the newly hatched
spiderlings, with the following differences.

1 Individuals which have the abdomen wider and deeper,
with the dorsum much more strongly arched and the pedicel
placed further back. In such individuals the abdomen is almost
always distinctly larger. These are females.

2 Individuals which have the abdomen narrower and less
deep, with the dorsum only moderately arched or not infrequently
flattened or even indented. Such individuals have in almost all
cases the abdomen smaller. These are males.

2 2 yj

i IIe ie
ee) ea
OC ier lesmen
Cee le ee
Oe Ge 1a er

Fic. 2

Intermediates do not occur between these two groups. In Fig.
2 I have illustrated such differences by showing in outline the
abdomina of spiderlings from several cocoons. ‘To the left is the
number of the particular cocoon, and on a line with it outlines of
the abdomina of the largest and smallest females, and of the largest
male of that cocoon, each abdomen shown on lateral and ventral
view. It is hardly necessary to add that plane drawings cannot

438 Thos. H. Montgomery, te

represent these differences as clearly as the rounded originals do.
Now these are like the form and size differences of the abdomina
of the adults; the adult female has the more arched abdomen with
the pedicel placed further back, while the male has the narrower
and flatter abdomen with the pedicel situated further forward.
Therefore I conclude, and no reasonable objection can be enter-
tained to this opinion, that those spiderlings most resembling in these
particulars the adult females are females, and those most similar
to the adult males are males. And it will be recalled that inter-
mediate individuals are not found, and one can separate rapidly
and unhesitatingly the spiderlings from a given cocoon into two
lots.

I had hoped to be able to distinguish the sexes at a still earlier
stage of growth, by constant differences in egg sizes, and for this
purpose preserved the eggs (all at the age of about twenty-four
hours) of the cocoon series of five different spiders. But this
expectation was not realized. The eggs in any cocoon differ
somewhat in volume, not greatly, but these size differences form
a graduated series and not two unbridgable groups. The sexual
differences of the hatched spiderlings being constant only with
regard to form but not always with regard to size of the abdomen
explains why we do not find female eggs always larger and so dis-
tinguishable from male eggs.

The following table represents the proportions of the sexes
in those cocoons of which the young were preserved shortly after
hatching. The first column gives the number of the mother and
the letters designating her successive cocoons. ‘The second gives
the day of oviposition, and the third the time interval between
oviposition and hatching. The succeeding columns state the
number of unhatched eggs, of males, of females, and the male
ratio: under the male ratio | understand the quotient obtained
by dividing the number of males by the number of females.

Sex Ratio of an Arnead 439
TABLE I
Cocoon Oviposition gine i Utes fon g ratio
hatching eggs

2000 days

A April 11 41 18 333 39 8.5
B May 5 32 19 228 57 4.
Cc 25 25 II 270 75 3-6
D June 5 20 ° 147 121 1.2
E 12 20 71 247 47 5.2
F 22 19 10 140 95 1.4
G July 1 19 5 | 132 165 8
H 8 19 II 146 102 1.4
I 16 19 42 145 94 1.5
if 25 19 136 92 69 1.3
K Aug. 4 18 63 60 76 i)
2002

A April 14 48 193 5) 71 09
B May 3 31 ° 280 27 10.3
Cc 21 25 ° 243 33 73
D 31 22 16 208 53 3-9
E June 9 19 9 186 68 2.7
F 15 20 I | 261 25 10.4
G 23 19 9 292 21 13.9
H July 1 20 4 325 20 16.2
I 8 20 RB | 242 18 13.4
J 20 19 I | 176 15 Tl. 7,
2003

B 404 2 202.
Cc April 15 40 2 300 46 6.5
D May 8 33 ° 301 9 33-4
E 23 25 ° 355 20 19 /89/
13 June 3 21 ° 313 37 8.4
G 10 20 ° 319 18 17.7
H 17 21 ° 331 12 27715
I 25 20 ° 316 17 18.5
J July 2z 20 ° 493 18 27.4
2008

A 239 6 39-8
B April 15 40 222 13 47 2
Cc May 6 33 ° 256 38 6.7
D 20 25 ° 246 25 9.8
E 29 22 ° 309 22 14.

440 Thos. H. Montgomery, fr.
TABLE I—Continued
Cocoon Oviposition pcre Unhatched | fot 2 ratio
hatching eggs |
|
2008 days | |
F June 7 19 3 367 31 11.8
G 12 19 ° 355 27 13.1
H 18 | 20 ° 250 50 ine
I 25 | 19 | oC 307 28 10.9
il July 1 19 | 2 287 40 Wied
K 7 19 60 | 237 15 15.8
2012 |
A 8 526 93 5.6
2013
A 646 6 107.6
B April 11 41 | 33 577 48 12.
€ May 7 32 3 316 15 21.
D 24 26 I 430 9 47-7
E June 4 21 I 404 20 20.2
EF 12 20 ° 398 10 39.8
G 23 20 I 432 12 36.
H 30 20 3 319 16 19.9
I July 16 20 3 432 12 36
J 26 20 2 282 9 31.3
K Aug. 5
2014
A 525 I 525.
B April 15 38 30 284 34 8.3
iC May 13 29 I 366 19 19.2
D 28 23 ° 474 24 19.7
13; June 8 20 o 448 40 TIe2
F 17 20 I 393 ef) 39-3
G 26 20 5 395 24 16.4
H July 2 20 6 432 22 19.6
I 12 19 2 367 9 40.7
J 24 19 3 328 16 20.5
2015
A ° 519 27 19.2
B May 6 32 ° 419 15 27.9
(e 23 25 ° 507 21 24.2
D June 3 20 ° 441 22 20.
E 12 20 ° 440 4 110.
F 22 20 ° 405 18 22.5

Cocoon

2015

[rate Mae? 0 |h:(e9)

2029

PAS KY POR wAeaAw

azamoaOw?

Oo4ZzZ4RAGHH mots
na

Oviposition

July 1

Aug. 6

Aug. 7

April 11

May 19
June 13

Sex Ratio of an Aranead 441
TABLE I—Continued
Time to Unhatched a 9 Siratio
hatching eggs

days
20 I 486 17 28.5
20 I 399 8 49-8
19 ° 348 14 24.8
18 ° 316 5 63.2
18 I 276 14 19.7
40 I 286 33 8.6
37 3 278 29 9-5
pe) 3 ZO], 49 4-2
25 | 5 280 45 6.2
23 18 172 59 2.9
20 10 193 21 9.1
20 6 191 21 9.
21 121 59 42 1.4
20 76 105 35 3.
28 143 10 ° 10.

32 2 208 32 6.5

399 16 24.9

40 23 533 7 7-5
25 4 311 37 8.4
20 4 287 73 3-9
21 ° 311 40 Fe
20 5 204 50 73
20 21 214 94 2.2
19 3 282 89 3-1

| 19 I 210 72 2.9

| 17 3 42 72 .58
41 I 643 25 25.6
36 3 387 18 215
26 ° 434 45 9-6
20 4 412 58 7.1
19 3 429 18 23.8

Thos. H. Montgomery, fr.

TABLE I—Continued

442
Cocoon Oviposition
2021
G June 22
H 30
I July 9
J 18
K 30
L Aug. 6
M Sept. 25
2022
A
B May 6
c 24
D June 3
E 10
F 17
G 25
H July 2
I 10
J 19
K 27
L Aug. 8
2023
A April 27
2030
A
B May 13
2031
A
B April 16
& May 11
D 25
3} June 4
F II
G 18
H 26
I July 2
yi II
K 18
L 28
M Aug. 24

Time to
hatching

days
19

Unhatched
eggs

» On W >A

OrIAnNwW FAN Ff

imal
i

332
392

194
265

118

9 G ratio
24 16.2
33 Io.

16 23-3
Io 39-2
56 3+4
45 537
6 98.

10 24.7
31 TIT
28 Te4
51 4.8
32 8.4
32 9.2
29 IZ0E
24 10.6
44 532
26 9-5
52 12
96 1.2
7 58.5
42 4-7
61 12
47 6.2
60 2.8
28 39
50 -98
9 1.6
33 13

Sex Ratio of an Aranead 443

Notes to Table I

Where a blank is left in the column headed ‘‘oviposition” it indicates that the cocoon was found
when the mother was captured, the day of oviposition therefore unknown; where a blank is left in the
column ‘‘Time to hatching” it signifies either that this was not determined, or else that the eggs did not
hatch; where a blank occurs in the column ‘“‘unhatched eggs” it signifies that these were not counted,
while a blank in the remaining columns indicates that the eggs did not hatch. Certain cocoons need
further explanation, as follows:

2002K. Made September 18, too late to te entered into this table; it did not hatch.

2003A. Proportion of the sexes not given because many escaped at hatching.

2012A. A wild cocoon the mother of which was not secured.

2013K. Eggs killed by mold, the only such accident.

o16A,B. Cocoons hatched when found, perhaps made the preceding season.

2016C. Found April 6 and hatched April 17, but the bottle of young dried up so that they could
not be counted.

2017. Other cocoons of this spider were used for the egg series.

2020A. A wild cocoon that proved to be parasitized.

2021A. A wild cocoon found empty.

2023, 2030. Other cocoons of these spiders were used for the egg series.

In the succeeding table these data are so summarized as
to bring out the reproductive differences of the several spiders
entered in the previous table, the “totals” of the third and seventh
columns being averages.

TABLE II
1 | Average no. | |

Spider | No. of cocoons |of obec) Unhatched 5; 9 Average

that hatched eggestoa | eggs o ratio

cocoon

2000 II 35 386 1.940 940 2
2002 | 10 23.6 36 2,220 351 6.3
2003 9 22 2 39132 179 17.
2008 II 26. 287 2,866 329 8.7
201z I 8 8 526 93 5.6
2013 10 4-7 47 4236 157 26.9
2014 I0 5. 50 4,012 19 20.1
2015 II 27 3 45556 165 27-5
2016 10 44.4 444 1,681 334 Ss
2017 I 2 2 208 32 6.5
2020 * 10 6.4 64 2,793 614 5
2021 II G52 59 4,246 348 12.2
2022 12 9.9 119 3,468 365 9-5
2023 I 42. 42 118 96 1.2
2030 2 ° ° | 609 49 12.4
2031 7 160.3 1122 | 599 288 ae
Totals | 127 22.6 | 2871 | 375210 4539 8.19

444 Thos. H. Montgomery, fr.

From these data we infer the following conclusions:

1 The average male ratio (number of the males divided by
the number of the females) is 8.19, determined from a count of
41,749 newly hatched spiderlings. Among the progeny of a par-
ticular female this ratio was never lower than 1.2 nor higher than
27.5. [oa cocoon the average number of hatched males is 292.9,
of hatched females, 35.7, and of unhatched eggs, 22.6; the average
number of eggs to a cocoon is 351.2. Of the total of 127 cocoons
entered in this computation, only 8 showed a male ratio of less than
one, and from only one (2016M) did only males emerge and
no females, this being the only “unisexual” cocoon. The highest
male ratio in any cocoon, excluding the case of 2016M just cited
was 202 (in 2003B); in eighteen cocoons the male ratio was 30 or
higher.

2 The objection might be raised that the above average male
ratio of 8.19 might not be the normal one for the species, but might
be induced by the life of captivity of the mothers. Therefore I
have considered separately the ratio in cocoons made in the natural
state and brought into my study to hatch out. Such cocoons are
the following of Table I: 2008A, 2012A, 2013A, 2014A, 2015A,
2020B, 2022A, 2030A, 2031A. These present a total of 3866
males and 223 females, giving the average male ratio of 17.3, con-
siderably higher than the ratio 8.19 obtained from the total of
cocoons [ raised. Whether this difference is due to difference in
the mode of life of the mothers, or rather so the fact (to be brought
out later) that the male ratio tends to be highest in the first cocoon
of a series, I cannot say. ‘These figures would show at least that
the high male ratio of captive cocoons cannot be ascribed to
artificial conditions, and indeed there is no reason for thinking
that the imprisonment of the mothers could affect this ratio.

3 It will be noticed that the male ratio was determined for
each cocoon, accordingly also for the average of all cocoons, from
the spiderlings that hatched out because I could not distinguish
the sexes before the time of hatching. ‘That is, the male ratio of
those eggs that did not hatch could not be ascertained, and this
is the single disturbing error in the above calculations. ‘Table I
furnishes the number of unhatched eggs for each cocoon, and

Sex Ratio of an Aranead 445

Table II the average number for each series, for the 127 cocoons
from which young emerged; 2871 eggs were infertile in the 127
cocoons from which hatched 41,749 spiderlings. Were those
undeveloped eggs all males, the male ratio would be increased to
8.8; were they all females, decreased to 5.1; yet there is no prob-
ability of either of these extreme cases. For when the male ratio
is unusually high (30 or higher) the number of unhatched eggs
to a cocoon is small, and where the male ratio is unusually small,
a good case of which is the series 2031 of Table I, the number of
unhatched eggs is generally but not always very high. ‘Therefore
it is probable that a large proportion of such undeveloped eggs
are males, and consequently the error introduced by such eggs is
probably a small one.

Next, as to the cause of lack of development of certain eggs.
Mechanical jarring of the freshly laid eggs of spiders has been
proved to be fatal to them ever since the observations of Herold,
so that the handling of the cocoons in the removal from the cage to
the hatching bottle may have prevented the development of some
eggs. Yet I believe the arrest of development was rarely so
induced, for I took great pains to handle all cocoons with extreme
gentleness and, as we see from the third column of Table II, the
average number of unhatched eggs to a cocoon varies with the dif-
ferent mothers which would not be the case were it due to acci-
dent. Probably, therefore, infertility of eggs is due to lack of
fertilization; and indeed the frequent happening of the last cocoons
of a series proving most infertile is to be ascribed to the supply
of spermatozoa becoming exhausted. ‘To test whether normal
parthenogenesis occurs | raised two immature females to maturity
without benefit of males; one moulted in March and the other in
April, which brought them to the mature condition with fully
formed epigyna, and both were allowed to live, with good feeding,
until August 30. One of them laid no eggs at all; the other made
a cocoon containing a few infertile eggs on June 18, and on June
26 dropped on the floor of the cage, without constructing a cocoon,
a mass of eggs that also proved infertile. “These two individuals
had not been impregnated, and all the eggs laid by them were

446 : Thos. Ef. Montgomery, vig

infertile, which renders unlikely the occurrence of parthenogen-
esis in this species.°

4. When we compare the male ratios of individual cocoons
given in the most right hand column of Table I no constant relation
is found between this ratio and the position of a particular cocoon
within a series. ‘The ratio may or may not be markedly different
in immediately successive cocoons as well as in cocoons at the
extremes of the particular series. The male ratio does not reg-
ularly increase from the first to the last cocoon, nor does it reg-
ularly decrease. Yet it will be noticed that of the twelve larger
series of cocoons of Table I, in seven of them the first made cocoon
of the series shows the highest male ratio (series 2000, 2003, 2008,
2013, 2014, 2020, 2022).

2 OBSERVATIONS ON OTHER ARANEADS

The only observer who has furnished detailed data upon the
sexual relations in spiders is Doumerc.® In the autumn of 1839
he captured a mature female of Theridion triangulifer and liber-
ated it in a room where it formed a web ina window frame. On
April 23 following it made a first cocoon from which only males
emerged and on May 10 a second cocoon from which hatched
only males. On June 16 she was impregnated by a male, on
June 26 made a third cocoon from which only females emerged
and on June 28 a fourth cocoon from which came out only males.
Doumere does not state how he distinguished the sexes of these
spiderlings; and for such “unisexuelliparous”’ species he con-
cludes that there are two uteri, one for males and the other for
females, and that oviposition does not take place from both at
the same time.

In collections adult females are usually more numerous than
adult males, due simply to the fact that males in their mature
condition are usually short-lived. But during the mating season
males are more abundant in most species, certainly among the

5 Compare Montgomery: On parthenogenesis in spiders. Biol. Bull., 1907.
® Notice sur les Cocons a pontes unisexuellipares de l’Aranéide Theridon triangulifer, Walck. Ann.
Soc. Entom., France. 9, 1840.

Sex Ratio of an Aranead 447

Argiopids, Theridiids and Lycosids. In genera like Filistata,
where the males are rare and where normal parthenogenesis
seems to occur, it may be that the male ratio is smaller than 1.

For Theridium tepidariorum C. Koch I found? a size differ-
ence or dimegaly of the eggs, and concluded it was probable that
females emerge from the larger eggs and males from the smaller
ones; I showed also that some cocoons contained only large eggs
and others only small ones, that therefore some may furnish only
females and others only males, and that usually one kind of egg
greatly predominates in number. Unfortunately this species is
not found at Austin so that I could not keep females to test this
point, and in my collection I have only a few bottles of newly
hatched young. ‘These specimens exhibit, however, two kinds of
individuals that do not intergrade; ones with larger and more
arched abdomina, others with smaller and flatter abdomina;
since these correspond respectively with the differences of the
adult females and males, I take them to be females and males,
respectively. Using this criterion I found the proportion of the
sexes of the young from four cocoons to be as follows:

TABLE II
Cocoon Unhatched eggs fou g co Ratio
; |
1058 numerous 3 112 | -03
1059 I 431 | ° | 431
1060 | ° 186 p 93-
|
1061 | 9 | 237 5 | 47-4

These examples are too few to allow any conclusions beyond
the one that in a particular cocoon one sex greatly predominates.
These relations are quite similar to those found by Doumerc for
T. triangulifer, a particular cocoon having a predominance of a
particular sex, but quite different from the relations in Latrodectus.

Therefore there is probably not a common male ratio for all
species of spiders.

7 Probable ‘dimorphism of the eggs of an Aranead. Biol. Bull. xii, 1907.

448 Twos, Jal Montgomery, Yt.

3 THE ORIGIN AND FIXATION OF SEX RATIOS

There are obviously two sides to the question of sex determi-
nation: the one, the process that regulates kind and succession of
the sexes of one offspring-unit (totality of offspring of one parent),
the other, the process of differentiation of the sexes and the origin
of sex ratios. The second question is essentially phylogenetic,
but if it should be proved that there is a distinct inheritance of
sex then this phylogenetic aspect will come to have an important
bearing on the other. We are now concerned with the question
of the origin of sex and of sex ratios.

The opinion is fairly general that in most animal species, those
exhibiting parthenogenesis excluded, there is no great disparity
in number between the sexes. ‘This has followed from a consider-
ation of the numbers of the sexes in man, for so far in no other
animal except the horse, is there available any computation of the
sexes based upon a count of large numbers of individuals at birth.
Only such computations have value, it is hardly necessary to add,
that are founded upon the count of the sexes at the time of birth
or earlier because the mortality after birth frequently varies with
the sex, the males in certain lower animals being more short-lived
and less resistant. We have found that in Latrodectus the male
ratio is 8.19, there being born more than eight males to every
female. For man, where the statistics are the only ones more
numerous than those of Latrodectus, the proportion of males to
females in 10,864,950 births is 1036 to 1000, the male ratio there-
fore 1.03, as taken from the compilation presented by Pike in his
Table III.* According to Morgan® for “‘still-born infants, fully
formed, but not alive,’’ Quetelet found 133.5 males to 100 females,
a male ratio of 1.33, and Bodio (from whose statistics I compute
an average) a male ratio of 1.31. Darwin?’ tabulates the births of
English race horses, 25,560 in all, giving 99.7 males to 100 females.
For Bufo lentiginosus King" found in individuals that had com-

8 Pike: A critical and statistical study of the determination of sex, particularly in human offspring.
Amer. Nat. 41, 1907.

9 Experimental Zodlogy. New York, 1907.

10 The descent of man, new ed., New York, 1886.

1 Food as a factor in the determination of sexin Amphibians. Biol. Bull., 1907.

Sex Ratio of an Aranead 449

pleted their metamorphosis 241 males to 259 females, a male
ratio of slightly less than 1; and other students have constated a
low male ratio in Amphibia. These examples, based on cases
where relatively large numbers of individuals were counted before
the age of maturity, and all with the exception of Bufo at or before
the time of birth, are sufficient to indicate that different species
have different sex ratios, and that the sex ratio may be a quality
of the species.

Now when there is a male ratio of 8.19 as in Latrodectus, such
a proportion of the sexes can be explained neither upon Newcomb’s
theory of chance” nor yet upon Castle’s idea of the Mendelian
inheritance of sex. Some other explanation is called for and,
as I shall proceed to argue, it is probably to be sought in the factor
of selection coupled with segregation.

In the first place the distinction of the sexes is a difference of
reproductive power. The female is the reproductive individual,
while the male is not reproductive but impregnatory, for females
can reproduce without males, as in cases of parthenogenesis, but
males are unable to reproduce of themselves. The male is dis-
tinctly the less important organism for the perpetuation of the
race. ‘There is no reasonable proof for the formula of Geddes and
Thompson" that the male is more katabolic and the female more
anabolic, for that is merely an unfounded statement. The sexual
difference is one of degree of reproductive ability.

Probably in the earliest racial species all individuals reproduced
in equal measure; this is probable simply because sexual dimor-
phism implies a rather advanced differentiation, therefore one
that should have developed more or less gradually. ‘The origin of
sex 1s most easily concerved in the following manner. Indi-
vidual variation within a species affects so far as we know every
quality, therefore reproductive ability would be a quality subject
to variation. Among the fluctuants of a species in which sex had
not yet become pronounced there would be a series of individuals

” A statistical inquiry into the probability of causes of sex in human offspring. Carnegie Inst.
Publ. 11, 1904.

8 Castle: The heredity of sex. Bull. Mus. Comp. Zool. Harvard, 1903.

4 The evolution of sex. London, 1897.

450 T hos. Ee Montgomery, hfs

extending from such with the greatest to such with the least repro-
ductive ability; the former would be incipient females, the latter,
incipient males. With this difference in reproductive ability
would certainly be associated metabolic differences, indeed the
latter would probably occasion the former.

Even racially older than observable sex difference is the process
of conjugation, which at the start had no immediate connection
with reproduction. Conjugation has been fixed by selection in
that it aids the race by strengthening the reproductive individuals
and making them more efficient in generation, conjugation being
in certain Protozoa a form of nourishment.

Conjugation being then of use to the race by strengthening or
stimulating the reproductive individuals, selection would preserve
those segregations of individuals in which variation with regard
to reproductive ability is most marked, for in such species con-
jugation would be most effective by occasioning the most diverse
substance intermixture. Selection coupled with segregation
would in time tend to eliminate the means, as the least useful in
conjugation, and to preserve those individuals most dissimilar in
reproductive capacity. This would terminate in a group of repro-
ductive individuals, females, and of fertilizing individuals, males,
not connected by intermediates.

With regard to the sex ratio of a particular species, Pike (/.c.)
concludes that it may “be looked upon as one of the physiological
adaptations of the species, determined by the conditions of its
existence. * * * If sex is hereditary, we might reasonably expect
that the relative numbers of male and female births in any species
would be those which, after deducting the early deaths, would
confer upon the species at the period of sexual maturity of its
individuals the greatest advantage in the struggle for existence
so far as the production of young is concerned.”” This explanation
is to my mind entirely just, and the factors would be, to carry the
idea out further than Pike did, those of selection and segregation.
Those species continue that survive in the struggle for life, and
this struggle is the endeavor to insure offspring. Selection oper-
ates by removing those species whose reproductive ability can-
not successfully meet this struggle. Therefore selection would

Sex Ratio of an Aranead 451

preserve, ceteris paribus, those races in which the females are
either most reproductive or else most caretaking of their young,
and in which there is at the same time a sufficiency of males to
insure the needed fertilization of the eggs. Within a given species
the male ratio would be subject to individual variation: some
females would produce a preponderance of males, others of females.
In the breeding area of such a species different groups of individ-
uals would come to show different male ratios, just according to
the productive peculiarities of their females, and in agreement with
what we understand of the action of segregation or physiological
selection in general. There would be groups with an unneces-
sarily large male ratio, others with the male ratio injuriously
small, others with the male ratio just rightly proportioned to the
number of females to be impregnated. An excessively high male
ratio would be a waste of males, and too low a male ratio a waste
of eggs because then all the eggs could not become fertilized; in
both these cases there would be an overplus of individuals that
would not be of service in procreation. Accordingly, selection
would preserve such groups of individuals in which the male
ratio is most nicely proportioned, most closely proportioned, to
the number of females needing to be impregnated. It would pre-
serve them because they would leave the most offspring. The
other segregations of individuals would become eliminated because
they include a waste of energies and individuals. Selection and
segregation would certainly be efficient factors, while it is more
doubtful whether heredity would also play a part.

What the male ratio would be in a particular species would
vary with different conditions, and particularly with differences
in the mode of life of the sexes. Where the sexes are most alike
in general habits of life, where internal impregnation of the female
is necessary and where the male cannot impregnate more than one
female, the proportion of the sexes would be most equal. Where
the males are physically quite as strong or even stronger than the
females, and where the male has the habit of impregnating several
females, it might be that the male ratio would sink below 1;
whether polygamous gregarious species should be reckoned here we
cannot say offhand, for the number of males born should be higher

452 T hos. H. Montgomery, Fr.

than the number that mate, seeing that many may be killed by
direct competition. More generally the male lives a simpler life
than the female, is less active both physically and psychically,
less fit for the struggle for existence, such as is the male in the spi-
ders we have been considering; in such cases many males die
before reaching maturity, and for such species the male ratio would
be high. Then where eggs do not require fertilization, as in par-
thenogenetic generations, selection would remove the males.

Thus the average male ratio of a particular species would be
fixed primarily by selection and segregation: these factors would
confine in rather narrow bounds the ratio of that particular species.
They would keep the number of males nghtly proportioned to
the number of ova that are to be fertilized, without unnecessary
waste of either.

And since the factor of chance and the factor of Mendelian
inheritance cannot explain certain specific sex ratios, it is at least
suggested that these factors may also fail to determine sex within
the offspring unit.®

3Tt may have some statistical value to append a count of 8796 adult individuals of the Rose
chafer, Macrodactylus subspinosus, that I made during June, 1901, on individuals collected from
one small garden near West Chester, Pa., and which gives a male ratio of 1.31:

Date of 2
June 20 824 638
21 642 683

24 1568 1135
25 678 474

27 623 432

28 65 5

4 445

Totals 4989 3807

THE CHROMOSOMES IN DIABROTICA VITTATA,:
DIABROTICA SOROR AND DIABROTICA 12-PUNC-
TATA

A CONTRIBUTION TO THE LITERATURE ON HETEROCHROMO-
SOMES AND SEX DETERMINATION

N. M. STEVENS

With Turee Prates

In Publication No. 36 of the Carnegie Institution of Washington,
the spermatogenesis of a number of Coleoptera was described,
and discussed with reference to the determination of sex. The
study of the Diabroticas was begun at Cold Spring Harbor in the
summer of 1906, and I wish to express my gratitude to Dr. C. B.
Davenport for the privileges granted me both at the Carnegie
Institution for Experimental Evolution and in the research labora-
tory of the Brooklyn Institute. I am also much indebted to Miss
Isabel McCracken of Stanford University for material of Dia-
brotica soror, prepared with the greatest care and sent to me in
December, 1906, and March, 1907.

The same methods were used as in previous work on the germ
cells of the Coleoptera. The germ glands were fixed in Gilson’s
mercuro-nitric fluid, in Flemming’s strong chromo-aceto-osmic
solution, and in Hermann’s platino-aceto-osmic fluid. Sections 5
thick were stained with iron hematoxylin or with thionin. The
aceto-carmine method was used for long series of Diabrotica soror
and Diabrotica 12-punctata.

DIABROTICA VITTATA

In the majority of the Coleoptera previously studied (85.7 per
cent), an unequal pair of heterochromosomes was found. ‘The
Diabroticas have an odd or unpaired heterochromosome, resem-

Tue JourNAL or EXPERIMENTAL ZOOLOGY, VOL. V, NO. 4

454 N. M. Stevens

bling in this respect the Lampyride and Elaterid as well as many
of the Orthoptera and Hemiptera.

In the spermatogonial equatorial plate of Diabrotica vittata,
we find 21 chromosomes (PI. I, Fig. 1) of various sizes and shapes.
If x be considered the heterochromosome, the others can be mated,
forming ten equal pairs. In sections stained with iron hamatoxy-
lin the division of the testis into several definite regions is very
striking. The resting spermatogonia hold little of the stain,
while the chromatin of the spermatocytes in synizesis and synapsis
stages is very black, and again the spireme stage is pale. The
synizesis stage here, as in several other Coleoptera (Stevens ’06),
appears to be a prolonged telophase of the last spermatogonial
mitosis. Fig. 2 shows the appearance of the short, crowded
chromatin loops in synizesis. Following this stage comes a period
in which the chromosomes are uniting in synapsis, and one finds
many nuclei similar to Fig. 3, some of the loops still short as in
Fig. 2, others longer and showing a sharp angle or a knob at the
point of union of two chromosomes. ‘There is no such definite
bouquet stage as in many forms, but one next finds a stage in which
there are irregularly disposed loops with many free ends and some
sharp angles like those in Fig. 3 (Fig. 4). In this stage the hetero-
chromosome (x) is for the first time evident, condensed against
the nuclear membrane. ‘This stage rapidly goes over into the
spireme stage (Fig. 5), where all of the chromosomes except the
heterochromosome (x) seem to be united into a single spireme
thread, and the points of union are no longer visible. The spireme
is very pale and the heterochromosome therefore very conspicuous.
There is nothing unusual in the prophases of the first division.
The spireme segments and splits longitudinally, the daughter
elements separate as in Fig. 6, then unite again and form rods,
dumb-bells, V’s and rings (Fig. 7). The chromosomes in the
spindle (Fig. 8) are so attached to the spindle fibers that in meta-
kinesis they separate into their univalent components, and go to
the poles as short thick V’s which mass together but soon separate
for the second division without any definite rest stage. The
unpaired heterochromosome (x) is of course connected with only
one pole of the spindle and does not divide in this division. Fig.

The Chromosomes in Diabrotica 455

g is the equatorial plate with the heterochromosome (x) at a differ-
ent level from the other chromosomes. Equatorial plates of the
second division are shown in Figs. 10 and 11, the heterochromo-
some (x) appearing in Fig. 10, and not in Fig. 11. All of the
chromosomes divide in this division giving equal numbers of
spermatids and spermatozoa containing ten and eleven chromo-
somes, respectively. The spermatids (Figs. 12 and 13) contain
a chromatin nucleolus (7), which is certainly not the heterochro-
mosome, since it is found in all of the spermatids. As the head
of the spermatozodn becomes more and more condensed, the
nucleolus gradually decreases in size and finally disappears (Figs.
14 and 15). The ripe spermatozoén has a very long slender
head (Fig. 16) which stains intensely black in contrast with
the earlier gray stages (Figs. 14 and 15).

DIABROTICA SOROR AND DIABROTICA 12-PUNCTATA

Diabrotica 12-punctata of the eastern United States and Dia-
brotica soror of the Pacific coast states resemble each other so
closely that one might easily be mistaken for the other. Both
are greenish yellow or yellowish green with twelve black spots
on the elytra. Kellogg describes Diabrotica soror as yellowish
green and Diabrotica 12-punctata as greenish yellow. The color
varies considerably with the age of the beetle. Diabrotica 12-
punctata averages larger,shades more on the yellow, and the under
side of the abdomen is green or yellow while in Diabrotica soror
itis black. The color of the abdomen seems to be the one exter-
nal character by which the two species can always be distinguished;
for the size, ground color, and size and fusion of spots are extremely
variable in both species.

A small amount of material of Diabrotica 12-punctata was col-
lected at Bryn Mawr, Pa., in October, 1906. On examining the
sections, it appeared either that the species was polymorphic as to
its germ cells, or that there must be two or more sub-species or
varieties, and possibly hybrids. It was too late to obtain more
material of this kind, so, through the kindness of Miss McCracken,
a supply of Diabrotica soror was secured for comparison with

456 N.M. Stevens

the eastern species; and in the summer and autumn ‘of 1907, 100
males of each species were studied by means of aceto-carmine
preparations. The character of the chromosomes in the male
germ-cells of the two species is precisely the same. About 50
per cent of the individuals examined have nine equal pairs of chro-
mosomes and an unpaired heterochromosome, while the remain-
ing 50 per cent have one, two, three or four additional small hetero-
chromosomes.

DIABROTICA SOROR

T ype I

The stages in the spermatogenesis of the first type are in most
respects similar to those of Diabrotica vittata. The sperma-
togonial metaphase has nineteen chromosomes (Fig. 17), the
unpaired chromosome (x) being the largest The synizesis and
synapsis stages are similar to those of Diabrotica vittata, but less
conspicuous in sections and the stages are less clear. The
changes that occur between the telophase of the last sperma-
togonial mitosis and the pale spireme stage (Fig. 18) prob-
ably take place much more rapidly in this species. A polar view
of the metaphase of the first spermatocyte division is shown
in Fig. 19, a lateral view in Fig. 20, and a late anaphase in Fig.
21. The odd chromosome is usually found at or near one pole of
the spindle in the metaphase (Fig. 20). The bivalents are similar
to those of Diabrotica vittata, and the first division separates
their univalent components. In preparations from Hermann
material the chromosomes of the daughter plates (Figs. 22 and 23)
often begin to show a vesicular condition and in telophase the
heterochromosome (x) forms a vesicle by itself, while the other
nine chromosomes are blended together (Fig. 24). Fig. 25 is
a later stage taken from a cyst in which some second spermatocyte
spindles were present, while Fig. 24 was from a cyst containing
a few first spermatocyte spindles. Half of the nuclei in these cysts
of course contain no heterochromosome. ‘The rest stage between
the two divisions is more pronounced than in Diabrotica vittata
where the chromosomes are simply massed together in telophase,

The Chromosomes in Diabrotica 457

and separate for the second division without the formation of a
nuclear membrane. The second spermatocyte equatorial plates
are shown in Figs. 26 and 27, the heterochromosome (x) appearing
in Fig. 26. All of the chromosomes divide in this division, giving,
as usual, two equal classes of dimorphic spermatozoa. ‘The
spermatids and spermatozoa are similar to those of Diabrotica
vittata. The chromatin nucleolus is found in the earlier stages
but is not visible in stages corresponding to Figs. 14 and 15, and
the head of the mature spermatozoén is only about one-half as
long.

T ype Ila

About two-thirds (33 out of 100 males collected at Mountain
View, Cal.) of the individuals belonging to the second type
have one additional small chromosome, making twenty in the
spermatogonia (PI. II, Fig. 28). Vhe additional chromosome
appears as a second heterochromosome in the growth stages (Fig.
29, s). In the first spermatocyte spindle the larger heterochro-
mosome (x) is found, as usual, near one pole of the spindle, while
the smaller one (s) may be in the equatorial plate (Fig. 30) or on
either side of it (Figs. 31, 32, 33), closely associated with x or as
widely separated from it as possible (Figs. 33 and 31). Fig. 34
is a polar view with the two heterochromosomes near one pole of
the spindle. “The small chromosome may or may not divide in
the first division. In some individuals it almost always (possibly
always) divides as in Fig. 35 later than the other chromosomes.
In other cases it may be found undivided between the daughter
plates (Fig. 36), outside of one of them (Fig. 37), or it may be
concealed in the general polar mass of chromatin. In the telo-
phase and brief rest stage (Figs. 38 and 39) it is often quite dis-
tinct from the remainder of the chromatin. Whether it divides
in this mitosis or goes undivided to one pole or the other seems to
be a matter of chance, depending perhaps on the part of the spindle
which it happens to enter in the prophase. It seems to be much
less automatic in its behavior than the other chromosomes. ‘This
peculiarly erratic behavior of the small heterochromosome in the

458 N. M. Stevens

first division gives, or may give, in the same individual six differ-
ent kinds of second spermatocytes with reference to this chro-
mosome (s), while there are, as usual, two kinds with reference to
the large heterochromosome (x). If the small chromosome goes
undivided to the same pole with the odd chromosome (x) (Fig. 33),
we have second spermatocytes containing nine and eleven chro-
mosomes (Figs. 40 and 41); if it goes undivided to the other pole
(Figs. 31 and 37), the resulting second spermatocytes each contain
ten chromosomes, one showing the large the other the small hete-
rochromosome (Figs. 42 and 43); while if it divides, the second
spermatocytes contain ten and eleven chromosomes (Figs. 44 and
45). As might be expected one finds two conditions in the second
spindle. Either a small daughter chromosome is found outside
of the equatorial plate (Fig. 46), or the small chromosome which
has not divided in the first division, divides in the second (Fig. 47).
Both conditions may be found in the same cyst. It is, of course,
in only a few favorable spindles that it is possible to see the small
chromosome actually dividing, but the metaphases are readily
separated into two classes, one where all of the chromosomes are
in the equatorial plate (Fig. 48) and another in which one small
chromosome, which from its form and size is evidently a daughter
chromosome from the first division, appears outside of the plate
and often quite near one pole (Fig. 46). It is therefore quite cer-
tain that the small heterochromosome divides in either the first
or second division but not in both. Clear daughter plates of the
second division have never been found.

The conditions described above lead to the production of two
equal classes of spermatozoa with reference to the large hetero-
chromosome (x) and four classes, which may be quite unequal,
with reference to the two heterochromosomes.

CG A
: ne variable numbers.

Equal numbers

(ASS SS)

II : i ‘ ithe ) variable numbers.

The Chromosomes in Diabrotica 459

If s always went to the same pole with x in the first division the
classes of spermatozoa would be as follows:

hae)
UII OR ect as

Equal numbers

If s always went to the opposite pole from x, we should get the
following results:

ss @) $B S
GL top mae

Equal numbers

If s always divided in the first spermatocyte division, there would
be four equal classes of spermatozoa:

I C ike } equal numbers.
Equal numbers +

} equal numbers.

A study of seventy or more individuals of this kind gives the
impression that the small heterochromosome most often divides
very late in the first division, but it 1s certain that there is consider-
able individual difference. In some cases nearly every anaphase
of the first division shows s dividing; in others, it is rarely or never
seen dividing in the first spindle, and as stated above, all of the
various possibilities have been found in one individual.

T ype IIb

Fifteen out of the same 100 males of Diabrotica soror had two
small heterochromosomes in addition to the eighteen ordinary
chromosomes and the large heterochromosome x. ‘These are
shown in a spermatogonial plate (Fig. 49). The three hetero-
chromosomes may also be seen in a growth stage (Fig. 50), a
prophase of the first division (Fig. 51), lateral and polar views of
the metaphase (Figs. 52 and 53) and an anaphase (Fig. 54).
Fig. 55 is an equatorial plate of the second division. When
two small heterochromosomes are present both may go to either

460 N. M. Stevens
pole of the first division spindle, one to each pole, or one or both

may divide as in Fig. 54. The resulting combinations in the
spermatozoa are as follows:

)
| :
\ variable numbers.

Equal numbers

ct |
eed ag variable numbers.
+x+a2s |

Type Ilc

Three out of the same 100 specimens had three small hetero-
chromosomes, as shown in Fig. 56, a growth stage, 57 and 58,
metaphases of the first division.

T ype IId

One individual had four such small heterochromosomes which
may be seen in Figs. 59-65, growth stages and first spermatocytes.
Here one may find all of the possibilities with respect to division
and distribution of the small chromosomes. The possible com-
binations in the spermatozoa are therefore as follows:

variable numbers.

o> N

—

OOO 0 O
+ 4
i)

+
aN
&

Equal numbers

variable numbers.

+++4+4+
es 4 &

The Chromosomes in Diabrotica 401

There were no spermatogonial plates of type IIc and Id which
could be counted, and in no case, though many ovaries have been
fixed and sectioned and others examined with aceto-carmine, has
it been possible to determine the number and character of the
chromosomes 1n the female.

DIABROTICA I2-PUNCTATA

Exactly the same conditions as to the small heterochromosomes
prevail in Diabrotica 12-punctata collected at Bryn Mawr, Pa.,
as in Diabrotica soror at Mountain View, Cal. Out of the
first 100 males examined in October, 1907, 51 had no small hete-
rochromosome, 35 had one, 11 had two, 2 had three and 1 had
four, while in Diabrotica soror the numbers for the five corre-
sponding classes were 48, 33, 15, 3, I.

A few figures only will be given for Diabrotica 12-punctata. As
in many other Coleoptera, spermatogonial equatorial plates in
which the chromosomes are well enough separated for accurate
counting are rarely found. ‘The one shown for Diabrotica soror,
type IIb, in Fig. 49, was drawn from an aceto-carmine prepara-
tion in which the chromosomes had been separated by pressure
on the cover-glass. Figs. 66 and 67 are spermatogonial plates of
Diabrotica 12-punctata, type | and type Ila, drawn from sections.
There is some overlapping here, but no doubt as to the number
in either plate. Growth stages for the five classes are shown in
Figs. 68, 69, 70, 71 and 72. The larger size of both nucleus and
chromosomes in Fig. 72 is due to its having been drawn with the
same power from an aceto-carmine preparation. ‘These figures
also serve to show something of the diversity of form of the odd
chromosome (x). When no small heterochromosome is present
it usually is nearly spherical (Figs. 18 and 68). Where one or
more of the small chromosomes are found, it is as a rule somewhat
elongated (Figs. 69 and 70), often irregular in form (Fig. 72), or
much elongated and bent in U-form. Whether this difference
indicates some influence exerted by the presence of the smaller
heterochromosomes, or marks the individuals containing the small
chromosome as a separate species is not at present clear.

462 N. M. Stevens

In both Diabrotica soror and Diabrotica 12-punctata the small
heterochromosomes are usually quite closely associated with the
larger one (x) in the growth stages, but this is by no means invari-
ably true. It is not at all unusual to find them separated in some
cells and in one individual it was noted that the two were more
often widely separated. (Figures to illustrate this have been
thrown out for lack of space.)

Fig. 73 shows a metaphase of the first spermatocyte from the
one individual of this species which had four small chromosomes.
Figs. 74 and 75 are anaphases from the same section, from an
individual with two small chromosomes, showing in one case (Fig.
74, 5, and s,) both dividing, in the other (Fig. 75) one dividing (s,)
and the other (s,) passing undivided to the same pole with the odd
chromosome (x). In general, these small chromosomes are remark-
ably uniform in size. One case, however, was found among the
aceto-carmine preparations where an unusually small one was
constant for the individual (Figs 76-78). This very small chro-
mosome was not found dividing in the first spermatocyte and it
could not be followed in the second division. In one cyst the
spireme was segmenting, later than usual, into dumb-bell shaped
bivalents (Fig. 77), as in Tenebrio molitor (Stevens ’05, Pl. 6,
Figs. 177-179).

As inthe other species of Diabrotica it has not been possible to
find favorable stages for counting the chromosomes in the female.
One may be able to do this by breeding the insects and working
with the tissues of the larva or pupa. Judging from similar cases
where the female number is known (for the Coleoptera, Elater I,
Fig. 229, Pl. 13, Stevens ’06, and Photinus pennsylvanicus, figures
not yet published; Anasa tristis and other Hemiptera, Wilson ’o5
and ’06; Peeciloptera, Fig. 283, Pl. 8 and Fig. 294, Pl. 9, Boring
07), we must suppose that the female number for Diabrotica
vittata is twenty-two and for Diabrotica soror and Diabrotica 12-
punctata, type I, twenty. Since the small heterochromosomes
seem to be as likely to go to the spermatozoa which receive the
odd chromosome (x) as to those which lack it, it would appear
probable that the conditions with reference to the small hetero-
chromosomes 1n the female are the same as in the male, and more-

The Chromosomes 1n Diabrotica 463

over it 1s perfectly possible for more than four to occur in either
male or female, as will be seen from the tables on p. 8.

DISCUSSION.

Sex Determination

For the present it is necessary to assume that the number of
chromosomes in the female bears the same relation to the number
in the male as in other cases among the Coleoptera and Hemiptera
where an odd or unpaired Hi eae enaorie is present in the
male. The division products of the unpaired chromosome pass
to one-half of the spermatozoa and these spermatozoa fertilize
the eggs which develop into females; while the spermatozoa
which lack the odd chromosome fertilize the eggs which produce
males. ‘This still seems to be as far as we can safely go in dis-
cussing the relation of the odd chromosome to sex determination.
This chromosome is uniform in its behavior in the three species
of Diabrotica, and it seems clear that it alone of the heterochro-
mosomes described can have any connection with the determina-
tion of sex.

T he “ Supernumerary” Chromosomes

The small heterochromosomes in Diabrotica 12-punctata were
first seen in some first spermatcoyte spindles by Miss Anne M.
Lutz of the Carnegie Institution of Experimental Evolution,
Cold Spring Harbor, more than two years ago, but the matter
was not followed up.

Prof. E. B. Wilson, in a recent communication (Science, n. s.,
vol. 26, no. 677, p. 870), has given the name “supernumerary”
chromosomes to certain additional heterochromosomes in Meta-
podius (Hemiptera), and perhaps that name is as good as any
other for the additional small heterochromosomes which appear
in variable numbers in about 50 per cent of random collections
of Diabrotica soror and Diabrotica 12-punctata. As in Meta-
podius the number of supernumeraries is constant for the individ-
ual. In Metapodius the supernumeraries are described as accom-
panying a pair of idiochromosomes with which they frequently
unite to form a compound element in the second spermatocyte.

464 N. M. Stevens

In the Diabroticas they are present with a larger unpaired hetero-
chromosome, and there is no evidence that they are ever united
with it. The most puzzling characteristic of the supernumeraries
in the Diabroticas is the fact that they may in the same individual
divide in either maturation division, and when two, three or four
are present, each one may divide in either spermatocyte division,
thus giving great diversity in the chromatin content of the sper-
matozoa. In Metapodius the supernumeraries are described as
dividing in the first division.

Occasionally, as in Figs. 64 and 73 two of the supernumeraries
seem to be paired in the metaphase of the first division, but this is
probably accidental, as it is not constant in any individual.

The only other known case among the Coleoptera at all resem-
bling this is that of the steel-blue flea-beetle, Haltica chalybea,
which has a large and a small heterochromosome which are often
widely separated in the metaphase of the first spermatocyte (fig-
ures not yet published). In the anaphase, however, the two
heterochromosomes are found between the two daughter plates,
and one goes to each second spermatocyte. This is merely a
case of late pairing and the distribution of the division products
of the two heterochromosomes to the spermatozoa is the same as
in other cases of an unequal pair of heterochromosomes.

The first lot of Diabrotica 12-punctata were dissected out and
all fixed together, so there was no opportunity to connect differ-
ences in the germ cells with differences in external characters of
the insects, if such existed. In the California material obtained
in December, 1906, and March, 1907, from Miss McCracken,
each beetle, after dissecting out the testis or ovary, was preserved
in alcohol, and later placed in the vial with its germ gland. In
the December lot there was a difference of 1 mm. in the length of
the Elytra, some measuring 4.5 mm., others 5.5 mm. All of the
smaller beetles had the odd heterochromosome only, the others
one supernumerary additional; and it was quite naturally sup-
posed that there might be two distinct species or varieties, one of
which had only the large unpaired heterochromosome, the other
an unequal pair of heterochromosomes. In the March lot, one
exception occurred—a small beetle had the additional small chro-

The Chromosomes in Drabrotica 465

mosome, and there were several individuals of intermediate size
which had one or more supernumeraries. In June, July and
August, 100 males of Diabrotica soror were studied in California.
The length of the elytron from origin to tip, measured to the
nearest fourth of a millimeter in a straight line—not over the curve
—was recorded for each beetle, the insects numbered and kept
for future reference. After the hundred had been collected,
measured and studied, they were arranged in series according to
nuclear type. It was at once evident that the insects of type I and
type II were about equally variable in size, and that all of the varia-
tions in fusion of spots occurred in each type. In fact no constant
difference in external characters could be detected which might
indicate two species. Thinking that possibly the variability in
size might be different in the early and late broods, two lots of
100 each were collected November 1 and December 1, 1907, and
measured without regard to the character of the germ cells. The
results are given in a table below. Meanwhile too males of
Diabrotica 12-punctata had been collected, measured, and the
testes studied in aceto-carmine in October, 1907. ‘This species
is somewhat less variable than Diabrotica soror, but the two types
with reference to the supernumerary chromosomes show the
same kind of variations.

The variability in length of elytra of the different species and
types is shown in Tables I and I1.

TABLE I

Diabrotica soror.

Length of elytroninmm...........| 3 |3-25| 3-5/3-75| 4 |4-25|4-5 14-75] 5 5.25 5-55-75
Lyfe WOGouceeacane oobbanuse 1 | 4 | 14 4/16] 2] 6 | 1 | | 48
Day ovismemeferrstsistctetetyetsterises | I | CFU) Gul ean) peal Mel re I eye! | 33
Tb 2h struc een meeceniarae s lees 2 | 6 2 |} | | 15
MEO Gecognoapdoonseonbee | I 2 | | less
INGLY io sessoonecuonobaedon | I | | leer

| | |
June; 23\toyAupusti7cieicl--ileyasre-1s leek ° 5 | 0° | 27 | 11 | 34 3 | 16 I | 2 | | 100
Novemberstastosaeaeecenisostren ret Ase he Gh || pS eye roller at) Sl Be || | 100
Decémberhin cyrtaernterteeertetes I 9 9 | 25 | 17 | 23 | 11 4 I | 100
Watals . steisotster-fotelsteisisterare einen I} oO} 6| 2} 43 | 35) 81 | 40 | 66 | | 7 I | 300

466 N. M. Stevens

TABLE II
Diabrotica 12-punctata

Length of elytroninmm.......... | 3 |3-25) 3-5/3-75| 4 [4-25] 4-5]4-75] 5 [5-25] 5-5]5-75
Ay PEWS OF Ssrereinre fats evatel acre evexa/otse | ale} 7 | 23 I 2) 51
UIE 00h an aah omOHASdAGcagen I 7) 4] 3 a7 I 35
Ube azysmemisctiecietsh tasters ates I I 9 II
MUCH ENS oecoshOH OSs OAbOnOp I | I 2
EAS ab serereineete niente mcreracere| | | ee I

r | | |
pL Otale eres sietetors ehesets e1< (eit asvieisves= I Di] 2 rg) orgie ral Siltaulliaro

It will be seen from the tables that Diabrotica soror is somewhat
more variable and averages smaller in early summer than in late
autumn; also that there is a possibility of two or three intergrading
groups. The latter fact would not, however, seem to have any
significance with reference to the supernumerary chromosomes,
since in Diabrotica 12-punctata (Table II) the curve of variability
is very steep with one mode at 5 mm. ‘The t00 specimens of
Diabrotica 12-punctata included in Table II were collected on some
late goldenrod in one corner of a field on October 3, 4 and g; the
first 100in Table I, in one rose garden in small collections extend-
ing over about six weeks. In both lots, most of the insects had
recently emerged, and the conditions of temperature and nutrition
under which they had developed could not have varied very
greatly.

The one significant result so far as the supernumerary chromo-
somes are concerned is the parallel series of numbers for the five
types of the two species—Diabrotica soror, 48, 33, 15, 3, I and
Diabrotica 12-punctata, 51, 35, 11, 2, 1. Were it not for this par-
allelism of results in the two similar but geographically widely
separated species,tone might suppose the presence of the super-
numeraries to be accidental, due perhaps to an irregularity in the
breaking up of the spireme or to imperfect metakinesis some-
where in the history of the male or female germ cells. The behav-
ior of the supernumeraries in the growth stages of the spermato-

1 Diabrotica 12-punctata occasionally ranges into California, but belongs more especially to the eastern
half of the United States, being perhaps most abundant in the Mississippi Valley.

The Chromosomes in Dtabrotica 467

cytes would suggest that they might have originated in a detached
portion of the odd chromosome (x), but such a supposition is not
borne out by their later behavior in the maturation divisions, nor
is there any evidence of an unequal pair among the other chromo-
somes indicating accidental separation of a part of one chromo-
some.

The only evidence I have that the supernumeraries might be
chromosomes in the process of development or degeneration is the
one individual (Diabrotica 12-punctata, No. 83 of the lot of 100
collected in October, 1907) in which one very small supernumer-
ary was observed (Figs. 76-78). In other cases there seemed to be
remarkable uniformity in size without regard to the number
present.

If at some period in the past history of the race before the eastern
and western species separated one supernumerary arose in any
way, its peculiar habit of division, sometimes in one, sometimes in
the other maturation division, may have given rise to the propor-
tional numbers of the different types in the two species. Or it
may still be possible, as was surmised earlier in the study, that
(1) there will prove to be two distinct types (varieties or species) in
each of the present species, one having the large unpaired hetero-
chromosome only, the other having an unequal pair of hetero-
chromosomes like that in Haltica, and that (2) the irregularities in
time of division and the consequent peculiarities in number and
distribution of the supernumeraries in Diabrotica are to be attrib-
uted to hybridism. If this should prove to be true it would indi-
cate little or no hereditary value for these supernumeraries or for
the smaller members of the unequal pair in other Coleoptera. A
careful biometrical study of several external characters may bring
to light some differences which can be associated with the pres-
ence or absence of the supernumeraries. The only other differ-
ence in the chromosomes of the two types seems to be a varia-
tion in the form of the odd chromosome (x). In type I it is
usually nearly spherical in growth stages, while in type II it is
more or less elongated.

Until the material is investigated further, it hardly seems worth
while to discuss at any greater length the hereditary significance of

468 N. M. Stevens
3

the supernumerary chromosomes or the possible results of their
irregular distribution. It however seemed advisable to publish
the results which have been obtained, as considerable time must
elapse before more material can be worked over; and it is to be
hoped that another summer’s work in California with breeding
experiments and collections from different localities may furnish
the data which are now lacking, and clear up the whole matter.

SUMMARY

1 Dhiabrotica vittata has twenty-one chromosomes, ten pairs
and an unpaired heterochromosome which behaves like the odd
chromosome in other Coleoptera and in the Orthoptera and
Hemiptera homoptera, dividing in the second spermatocyte divi-
sion, but not in the first. Synapsis occurs at the close of the syni-
zesis stage. A chromatin nucleolus is present in all of the sper-
matids.

2 Dhiabrotica soror and Diabrotica 12-punctata both have in
all cases nineteen chromosomes, nine pairs and an unpaired hetero-
chromosome, which divides like that in Diabrotica vittata.
About 50 per cent of the individuals examined have only nineteen
chromosomes, the remaining 50 per cent have from one to four
additional or “supernumerary” chromosomes which divide in
either spermatocyte division, not in both, and may therefore give
rise to from four to ten different kinds of spermatozoa with refer-
ence to their chromatin content, in the same individual. The
percentage of individuals containing no supernumerary chromo-
some, one, two, three, or four supernumeraries, is nearly the same
for the two species—48, 33, 15, 3, 1 for Diabrotica soror at Moun-
tain View, California, and 51, 35,11,2,1 for Diabrotica 12-punctata
at Bryn Mawr, Pa. It has not as yet been possible to associate
the different nuclear types with variations in any external char-
acter.

Biological Laboratory of Bryn Mawr College
Bryn Mawr, Pa.

Nore—A part of the facts concerning the chromosomes in Diabrotica soror were given at the Inter-
national Congress of ZoGlogists in Boston, August 21, 1907.

The Chromosomes in Diabrotica 469

LITERATURE CITED

Borine, A. M. ’07—A Study of the Spermatogenesis of ‘Twenty-two Species of the
Membracida-, Jassida, Cercopide and Fulgoride. Journ. Exp.
Zo6l., vol. 4, p. 469.
Srevens, N. M. ’o5—Studies in Spermatogenesis with Especial Reference to the
‘Accessory’? Chromosome. Carnegie Inst., Wash., Pub. 36.
‘o6—Studies in Spermatogenesis. II. A Comparative Study of the
Heterochromosomes in Certain Species of Coleoptera, Hemiptera
and Lepidoptera, with Especial Reference to Sex Determination.
Carnegie Inst., Wash., Pub. 36, II.
Wiuson, E. B. ’o5—Studies on Chromosomes. II. The Paired Microchromo-
some, Idiochromosomes and Heterotropic Chromosomes in the
Hemiptera. Journ. Exp. Zodl., vol. 2, p. 507.
‘o6—Studies on Chromosomes. III. Sexual Differences of the Chromo-
some Groups in Hemiptera, with some Considerations on Deter-
mination and Inheritance of Sex. Journ. Exp. Zodl., vol. 3, p.t.
o7—The Supernumerary Chromosomes of Hemiptera. Science, n. s.,

vol. 26, p. 870.

DESCRIPTION OF PLATES

Figs. 1 to 48, 50, 54 and 66 to 71 were drawn from sections with 2 mm. obj. and 12 oc.; Figs. 49, 51
to 53 and 55 to 65 from aceto-carmine preparations with 2 mm. obj. and 6 oc.; Figs. 72 to 77 from aceto-
carmine preparations with 2 mm. obj. and 12 oc. The magnification of all of the figures was then
doubled with a drawing camera, and the plates reduced one-half.

Lettering used on Plates

x = the unpaired, “odd” or “‘accessory”” chromosome.

n = the chromatin nucleolus of the spermatids.

s =a “supernumerary” chromosome.

Sty 525 53, S4 = I, 2, 3 OF 4 supernumerary chromosomes in the same individual.

Prate I
Diabrotica vittata

Fig. 1 Spermatogonial metaphase, twenty-one chromosomes.
Fig. 2 First spermatocyte, synizesis stage.

Fig. 3. First spermatocyte, synapsis stage.

Fig. 4 First spermatocyte, postsynapsis stage.

Fig. 5 First spermatocyte, spireme stage.

Figs.6and7 First spermatocytes, prophase.

Figs. 8 and 9 First spermatocytes, metaphase.

Figs. 10 and 11 Second spermatocytes, metaphase.

Figs. 12 to 15 Spermatids.

Fig. 16 Ripe spermatozoén.

Diabrotica soror. Type I

Fig. 17 Spermatogonial metaphase, nineteen chromosomes.
Fig. 18 First spermatocyte, spireme stage.

Figs. 19 and 20 First spermatocytes, metaphase.

Fig. 21 First spermatocyte, anaphase.

Fig. 22 and 23 First spermatocyte, daughter plates.

Figs. 24 and 25 Second spermatocyte, rest stage.

Figs. 26 and 27 Second spermatocytes, metaphase.

THE CHROMOSOMES IN DIABROTICA PLATE I
N. M. STFVENS

xuy y
-wtee Xe
as se
On :
1
@.°
L)
= C73)
werd i
5 ¢ :
8
dns af =
syle" aime em oo es ae
10 a fe : :
15
WE pe
pee CSE Soe
: is 19
=
ee Ke
a) 00S ‘
of 00 16
ed se
22 23

Prate II
Diabrotica soror. Type Ila

Fig. 28 Spermatogonial metaphase, twenty chromosomes.

Fig. 29 First spermatocyte, spireme stage.

Figs. 30 to 34 First spermatocytes, metaphase.

Figs. 35 to 37 First spermatocytes, anaphase.

Fig 38 First spermatocyte, telophase.

Fig. 39 Second spermatocyte, rest stage.

Figs. 40 to 45 Second spermatocytes, metaphase, polar view.
Figs. 46 to 48 Second spermatocytes, metaphase, lateral view.

Type IIb

Fig. 49 Spermatogonial metaphase, twenty-one chromosomes.
Fig. 50 First spermatocyte, spireme stage.

Fig. 51 First spermatocyte, prophase.

Figs. 52 and 53 First spermatocytes, metaphase.

Fig. 54 First spermatocyte, anaphase.

Fig. 55 Second spermatocyte, metaphase.

THE CHROMOSOMES IN DIABROTICA PLATE II
N. M. STEVENS

Prate III
« Diabrotica soror. Type IIc

Fig. 56 First spermatocyte, spireme stage.
Figs. 57 and 58 First spermatocytes, metaphase.

Type IId

Figs. 59 and 60 First spermatocytes, spireme stage.
Fig. 61 First spermatocyte, prophase.

Figs. 62 to 64 First spermatocytes, metaphase.
Fig. 65 First spermatocyte, anaphase.

Diabrotica 12-punctata

Fig. 66 Spermatogonial metaphase, nineteen chromosomes.

Fig. 67 Spermatogonial metaphase, twenty chromosomes.

Fig. 68 First spermatocyte, spireme stage, no supernumerary.

Fig. 69 First spermatocyte, spireme stage, one supernumerary.

Fig. 70 First spermatocyte, spireme stage, two supernumeraries.
Fig. 71 First spermatocyte, spireme stage, three supernumeraries.

Fig. 72 First spermatocyte, spireme stage, four supernumeraries.

Fig. 73 First spermatocyte, metaphase, four supernumeraries.

Figs. 74 and 75 First spermatocytes, anaphase, two supernumeraries.
Figs. 76 to 78 First spermatocytes, unusually small supernumerary.

THE CHROMOSOMES IN DIABROTICA

PLATE III
N. M. Stevens
s >
2 x
ae eer we
<4 ae @ Or. 3S
a 3 © e° oS e = =x
d Af: C@eq « ee _ is 3
56 uet 58 a =
e---s
ce et @ = $ x x @

1° i ae ae >
19° : \ M

| | ENG
pee —

° 66
ex * 68

From the Department of Comparative Anatomy, Harvard Medical School, Boston, Mass.

THE EXPERIMENTAL CONTROL OF ASYMMETRY
AT DIFFERENT SEAGES IN GHE DEVELOPMENT
OF THE LOBSTER:

BY
VICTOR E. EMMEL

INTRODUCTION

The asymmetry of decapod crustacea has recently been studied
by Przibram (or, ’02, ’05, ’07), Morgan (’04), Zeleny (’05) and
Wilson (05). This asymmetry is manifest in the first pair of
claws or chela—one of which is larger and frequently structurally
different from the other. It has been found in some cases that
if the large chela is removed, a small one may regenerate in its
place. At the same time, the original small chela on the opposite
side of the body may grow into a large one. ‘This transposition
of chela is known as “reversal of asymmetry.” A complete
reversal of asymmetry follows the amputation of the large chela
in the adult of Alpheus as shown by Przibram and Wilson. On
the other hand, sucha reversal is not obtained in similar experi-
ments with the adults of the hermit crab and lobster as found by
Morgan and Przibram. This I have confirmed in regard to the
lobster by experiments with over 200 adults, in none of which was
there obtained a transposition of the chelz.

The previous studies have dealt only with adult animals. In
view of this fact it seemed desirable to investigate the establish-
ment of asymmetry at various stages in the growth of the lobster—
one of the forms in which reversal does not occur in the adult.
This has been done with the results about to be described.

The work was carried on at the Experiment Station of the Rhode
Island Commission of Inland Fisheries, and I desire to express my
indebtedness to Dr. A. D. Mead for generously permitting me to

Tue JourNar or ExperiMentaL ZoOLoGy, VOL. Vv, No. 4

°472 Victor E. Emmel

use the apparatus and the excellent material available at the

lobster hatchery.

NORMAL DEVELOPMENT OF THE CHELAE

A brief description of the normal development of the chelz
of the lobster may aid in understanding the nature of the present
experiments.

In the adult lobster, one of the two chele, either the right or the
left, is a rather long slender nipper claw, and the other is a larger
and more massive crusher. Each claw consists of a movable
jaw, the dactyl, and an immovable jaw or index. In very young
lobsters the right and left chelz appear alike. During the first
three larval stages they are embryonic in character. The claws
are relatively short and broad; the index is smaller than the dactyl
and both index and dactyl are beset with long hairs or bristles.
During the fourth and fifth stages, the claws have become long
and slender but are still alike. Characteristic tactile hairs and
pointed teeth appear and the claws now begin to resemble the
adult nipper type.

At about the sixth stage however a divergence in the differen-
tiation of the two chele becomes apparent. In one of the chelz
the nipper characters continue to develop. This claw retains
the long slender form characteristic of the adult. ‘Tactile hairs
are distributed in a dense fringe on each side of the dentate mar-
gin. The pointed cutting teeth are arranged in a linear series
for each jaw with the exception of a stout displaced tooth about
midway in the dentate margin of the index. In marked contrast
to this development of the nipper, the other claw becomes wider,
broad tubercle-like teeth develop, and the tactile hair of the nipper
type gradually disappears in successive moults. Thus the adult
crusher claw comes to be characterized by the almost entire absence
of tactile hairs, and the presence of broad crushing teeth; and bya
form larger and more massive than that of the nipper. The
final result is the establishment of the adult asymmetry.

In the development of the lobster therefore there is a series of
larval and adolescent stages, in which there is a transition from

Control of Asymmetry 473

symmetrical to asymmetrical chele. In this asymmetry the
crusher occurs as frequently on one side of the body as on the
other.

PLAN AND METHOD OF THE EXPERIMENTS

The present experiments were made in the following stages of
the lobster’s development—the stages being designated as first,
second, etc., according to the number of moults since the time of
hatching:

The second larval stage;

Fourth stage;

Fifth stage;

Twelfth stage or lobsters a year old;
Adult lobsters.

All of these experiments attempt to determine to what extent
asymmetrical differentiation of the chele can be controlled by
amputation. In lobsters, as is well known, an injured limb is
thrown off spontaneously or autotomously, separating along a cer-
tain “breaking plane” near the basal joint.

It was found necessary to exercise great care in the mutilation
and rearing of the delicate larval lobsters. The chela was
removed, under a small hand lens when necessary, by grasping
the tip of the limb with a pair of forceps. In the older lobsters
the chela promptly separates at the breaking plane. In the
younger lobsters however the separation is not so readily obtained
and a gentle pull may be required. The most difficult period in
which to keep the lobsters alive is during the second and third
stages. After several failures with ordinary aquaria the best
results were obtained by keeping the animals in a current of fresh
sea-water. This was accomplished by means of a rather elaborate
apparatus built in the pool of a wooden float. The bottoms of
pulp pails were removed and replaced by “bobbinet” cloth with
meshes small enough to prevent the escape of the lobsters. A
second cover, or false bottom, of mosquito bar was also found
necessary—not to confine the lobsters but to prevent the ever
present shrimp from pulling them out through the meshes.
These pails were then suspended in the water of the pool. In

474 Victor E. Emmel

each pail was placed a small paddle not unlike a boat propeller,
consisting of a vertical shaft with two horizontal blades at its
lower end. Each paddle was kept in motion by proper gearing
with a “live shaft.”” The blades were beveled so as to give an
upward movement to the current of water. In this way it was
possible to rear a small per cent of the mutilated lobsters through
the critical larval stages.

After the fourth stage, the lobsters were placed in a floating
car divided into small compartments. Each lobster was kept in a
separate compartment and a careful record made of its mutilations,
moults and regenerations.

EXPERIMENTS WITH LOBSTERS IN THE SECOND STAGE

On July 24, 1906, two groups of lobsters were. mutilated.
These lobsters had all hatched from the egg within about four
days. In Group A, the right chela, and in Group B, the left chela
was removed from each specimen. In spite of an exceedingly
great mortality, thirteen specimens were reared beyond the
sixth stage. After each moult the regenerated chela was inva-
riably removed. Thus the limb on the opposite side was given
a great advantage for growth in order to learn whether this chela
could be made to differentiate into a crusher. The results are
shown in the accompanying table. This table includes also the
data from a supplementary experiment made during the following
summer. In this experiment great difficulty was likewise experi-
enced in rearing the mutilated animals, for out of 200 larval
lobsters from which the right chela was removed, only three speci-
mens lived beyond the sixth stage.

From the data for these sixteen lobsters it will be observed that
when the chel had differentiated far enough to display asymme-
trical characters, the claws which regenerated after amputation
were all nippers; at the same time, the claws which were not
mutilated, being thus given the greater advantage in growth, were
all crushers.

Control of Asymmetry 475

TABLE I.

Larval lobsters in the second stage. Original chele symmetrical.
Group A Right chela removed

*In specimens Nos. 6 and 7, the regenerated right chela was not removed after the moult to the
sixth stage on July 14, and on account of unavoidable absence, a record of further moults was not kept.

} These specimens, unfortunately, died before there was a clearly developed asymmetry of the chelz.
In Nos. 4 and 5, the general appearance of the left claw and the characteristic double tubercle dentition
at the base of the jaws indicated that these claws were differentiating into crushers. No. 10 however
died on May 31 without having differentiated asymmetrically.

} It may be of interest to note that No. 15 was much slower in its differentiation than the other speci-
mens. At the close of the experiment in 1906, this animal showed no evidence of asymmetry. It was
kept through the winter and after three more moults during the following summer, this lobster, in
harmony with all the others in Group B, developed a crusher claw on the right chela.

LOBSTERS IN THE FOURTH STAGE

At the fourth stage the lobster has made a marked advance
toward the adult form. The chela however are still alike and
symmetrical.

476 Victor E. Emmel

On July 25, 1907, seventeen specimens were mutilated on the
day following the moult to the fourth stage. Each lobster was
mutilated by the autotomous removal of the right chela. The
results are shown in Table II.

TABLE II

Lobsters in the fourth stage. Original chele symmetrical. Right chele removed

H | |
| | |
| | | FINAL ASYMMETRY OF
No. | Stage Mutilation | Stage | Mutilation | Moult to CHEE
| | | Stage VI |- $<
| | Right Right
|
I | July 16 nipper | crusher
2 16 nipper | crusher
3 j 16 nipper | crusher
Ava| + |) eee ic 16 nipper crusher
5 2 va | z S 16 nipper crusher
6 4 ak 2 aa a, 16 nipper crusher
7 | & 5 = ze 16 nipper crusher
8 § 3 bee 4 16 nipper | crusher
9 = ta} E 3 (?)* nipper crusher
10 3 2 Mw 5 (?) nipper crusher
Il 2 2 & g, (?) nipper crusher
12 a a) 2 & (?) | nipper crusher
3 5 3 as = (?) | nipper crusher
14 3 re 3 a (?) nipper crusher
15 s s 4 (?) nipper crusher
16 | (?) | nipper crusher
17 @) nipper crusher
| | 1

* Specimens 9 to 17 moulted to the sixth stage a few days after July 16, but the date of moult was not
recorded.

It is readily seen that these results show a marked uniformity.

Without exception the mutilated claws became nippers; the claws
which were not mutilated became crushers.

LOBSTERS IN THE FIFTH STAGE

The fifth stage is especially important because, normally, at the
next moult asymmetrical characters are displayed. Consequently,
during this period there must be a rapid progress in the differen-
tiation of the chelz.

Control of Asymmetry 477

The lobsters were mutilated by the autotomous removal of the
right chele July, 1907. The mutilations were made about four
days after the moult to the fifth stage. Through the kindness of
Dr. A. D. Mead and his assistant, Mr. L. N. Wight, some of the
lobsters were kept alive until the final data could be obtained in
September. The results are shown in Table III.

TABLE II

Lobsters in the fifth stage. Original chele symmetrical. Removed right chela
four days after moult to fifth stage Fuly, 1907

FINAL ASYMMETRY, SEPTEMEER 21

No. :
Stage * Right chela Left chela
|

I seventh (?) nipper crusher
2 seventh (?) nipper crusher *
3 seventh ( ?) nipper crusher
4 seventh (?) crusher nipper

5 seventh ( ?) crusher nipper

6 seventh (?) crusher nipper

7 seventh ( ?) crusher nipper

8 died

9 aicd
10 died

* By September 21, these lobsters were apparently all in the seventh stage, although
the stage cannot be positively stated because a record of all the moults was not obtained.

These results are in marked contrast with those obtained for
the preceding stages. Instead of all the uninjured claws pro-
ducing crushers, they produced three crushers and four nippers.
At the same time, the regenerated claws, instead of being all nippers
include three nippers and four crushers. Since in the adult lob-
ster the crusher appears about as frequently on one side of the
body as the other (and this is equally true of the nipper), it appears
that the normal development was not modified by the removal of
one chela. Evidently therefore during the fifth stage, in which
the-chelz are apparently still symmetrical, the controlling influence
of such amputations upon symmetry disappears.

478 Victor E. Emmel

A point which should receive further investigation for this
stage is the time of mutilation with reference to the moult. It
will be observed that in the above experiments the mutilations
were made several days after moulting. In another experiment
the left chela was removed from a number of lobsters on the day
in which they had moulted to the fifth stage. Only four of the
specimens lived until the chele displayed asymmetrical characters,
but it is interesting to note that for each lobster, a crusher devel-
oped on the right or uninjured chela, and that the regenerated
claw was a nipper. ‘This result indicates that possibly the asym-
metry may be controlled at this stage, provided that the mutila-
tions are made sufhciently early.

IMMATURE LOBSTERS, A YEAR OLD

With the assistance of Mr. E. W. Barnes, superintendent of
the Experiment Station of the Rhode Island Fish Commission,
we succeeded in keeping about thirty-five lobsters, hatched in
July, 1905, until the following summer. At this time these year-
ling lobsters were all in about the twelfth stage and averaged two
inches in length. ‘The asymmetry of the chelz is clearly developed
at this age. But when it is recalled that the lobster does not attain
sexual maturity until about the fifth year, it will be readily appre-
ciated that these yearling lobsters were still quite immature and
at a period of rapid growth. It seemed desirable therefore to
ascertain the degree of stability which the asymmetry. may have
attained at this age as compared with the adult. The experimental
results obtained are shown in Table IV.

In this experiment on yearling lobsters, 15 were mutilated by
the autotomous removal of the crusher chele (Group A), and 14
were mutilated by the removal of both chele (Group B). In
both groups the regenerated claws were again amputated. In no
case in either group did these mutilations and consequent regen-
erations reverse the original asymmetry. Each yearling lobster
retained its original right or left handed arrangement of the
chelz.

It should be added however that in the case of the crusher

Control of Asymmetry 479

chela the regenerating claw is not always a characteristic crusher
from the outset, but frequently displays, at first, characters inter-
mediate between those of a crusher and a nipper.!

TABLE IV

Lobsters a year old. Original chele asymmetrical. Group A Crusher chela removed*

| RELATION OF CHELAE AFTER TWO
ORIGINAL RELATION OF CHELAE |

REGENERATIONS AND TWO MOULTS
LOBSTERS

Right Left Right Left

|
8 specimens crusher nipper | crusher nipper
: F | F
7 specimens nipper crusher nipper crusher

Group B Both chele removed*

8 specimens crusher nipper crusher nipper

6 specimens nipper crusher nipper | crusher

* After the first moult the regenerated chele were again removed from each lobster.

ADULT LOBSTERS

Przibram (or, ’02) and Morgan (’o4) have already observed
that in adult lobsters a typical reversal of asymmetry as the result
of amputation and regeneration of the chela has not been found.
In the course of my experiments over 200 adults were mutilated
by removing one or both chela. In no case did a crusher develop
on the side which had originally carried a nipper, and the same
was true, vice versa, for the nipper. As in the yearlings, but not
to the same extent, the regenerating crusher chela is not always
at first distinguishable as such, but may present characteristics
intermediate between the nipper and crusher (Emmel, ’06?).
Also in certain very rare cases, symmetrical chele of either the
nipper or crusher type may regenerate in place of the amputated
asymmetrical limbs (Emmel, ’06%, ’07?.) The fact to be empha-
sized however is that in these adult lobsters, the amputation of

1 Compare Emmel ’o6* and Przibram ’o7, p. 291.

480 Victor E. Emmel

neither one nor both chele produced a reversal of the original
asymmetry.

DISCUSSION

Until recently the phenomenon of reversal of asymmetry or
compensatory regulation, which Przibram and Zeleny found
Alpheus, was not supposed to occur in such forms as the lobster
or hermit crab. It appeared that these species were characterized
by a “direct regeneration” of the original asymmetry. But the
discovery that under certain conditions the adult lobster might
regenerate a crusher from the stump of the amputated nipper
Rhee (‘Emmel ’06?) demonstrated that in the lobster, at least, both
sides of the body might still retain the potentiality for the more
highly differentiated type of crusher claw. The present results
which show that asymmetrical differentiation can be controlled at
early stages of development in the lobster, suggest that similar
relations in the development and stability of asymmetry may be
found in other crustacea.’

Various theories have been advanced concerning the factors
which determine right or left asymmetry, and which may be dis-
cussed on the basis of the preceding experiments.

Herrick (’o05) studied the shrimp Alpheus, and concluded that
asymmetry of the chelz in Alpheus and also in the lobster “is
probably one of direct inheritance, all members of a brood being
either right or left handed. ‘That is to say, the normal position of
the toothed or crushing claw is not haphazard, but is predeter-
mined in the egg”’ (p. 225).

Conklin (’03, ’05), without discussing inheritance, shows how
inverse symmetry may be determined in the egg. He found rea-
son for believing that the cause of inverse symmetry, which occurs
regularly among some species and occasionally among all, man
included, is to be found in the inverse organization ofthe egg, and

?It is interesting to find this suggestion already anticipated by Przibram. In his important mono-
graph published in the Archiv. f. Entw.-Mech., Bd. 25, 1907, p. 310 (received while the present
paper was being written), he discusses the question,‘‘Is the possibility of the reversal of chela present in
those forms which have hitherto shown no reversal ?”” He concludes that in some of these (Callianassa

and Carcinus) the asymmetrical relations may be altered.

~*n
=

Control of Asymmetry 4

that this inverse organization may be due to the maturation of the
egg at opposite poles in dextral and sinistral forms (’05, p. 10).
On this basis, the alternate appearance of right or left asymmetry
in the lobster might be regarded as cases of inverse symmetry
“resulting from slight alterations in the localization of germinal
substances in the unsegmented egg.”

Morgan (’07) does not venture to decide between the possibili-
ties of “inheritance” and the structure of the egg, as determining
right or left handedness in various species. He says “both possi-
bilities seem to exist in the egg; but whether this can be referred to
alternate dominance and recession, or to purely local conditions
that arise during segmentation, is unknown” (p. 165).

It is evident that the present experiments at least demonstrate
that the asymmetry of the adult lobster is not necessarily inherited
nor even predetermined in the egg. However, the question still
remains as to what factors in normal development determine
right or left asymmetry. No evidence was found that the occur-
rence of right or left asymmetry in the lobster can be referred to
germinal units having “alternating dominance and recession.”
The fact that in early development a crusher can be produced on
either side of the body by the amputation of the opposite chela,
indicates that the factors which control asymmetry become opera-
tive after hatching. What these factors are, or how they may be
released by the amputation of a limb, is not known. We can
merely refer to the fact that in early stages of development of the
lobster the asymmetry of the chela can be experimentally controlled.
When asymmetry. has once been normally established, similar
experiments no longer reverse it.

That the accidental loss of a limb in the young lobster may
play an important role in determining the asymmetry of the adult
isnot improbable. For the autotomy of a chela during the exigen-
cies of moulting or as the result of injury, is a common occurrence,
especially among young lobsters. In an examination of several
thousands of fourth stage lobsters it was found that a large per
cent of the animals had lost either the right or left chela. In these
the right or left asymmetry would not be inherited or due to the
structure of the egg.

482 Victor E. Emmel

Przibram (’07),in his recent extensive work on “Die Scherenum-
kehr bei decapoden Crustaceen,”’ found experimentally that a
reversal of chelz could be obtained in six genera and eleven species
of crustacea, including forms in both the Macrura and Brachyura.
He then inquires whether this capacity for reversal of asymmetry
is a constant characteristic for a given species. Here he finds
that the statement that a reversal of asymmetry always follows
the amputation of the crusher (““K — schere”) in a crustacean
requires modification. For it appears that the readiness with
which reversal occurs varies inversely with the size of the animal.
Specimens of Athanas, Alpheus, Typton, Callianassa, Carcinus
and Portunus, which are under 10 mm. in carapace length,
showed a quick and complete reversal of asymmetry. But, on the
other hand, the larger specimens showed a decrease in the tendency
toward transposition of the chela, so that when the large chela is
removed the chela on the opposite side retains its original form.

It appears therefore that the relations found in the control of
asymmetry at different stages of development in the lobster, are
also true for other crustacea with asymmetrical chela. For both
in the lobster and in the forms described by Przibram, the various
stages in their development form a series beginning with a com-
plete control of asymmetry and ending with the disappearance of
such control. In other words, the possibility for experimental
control and reversal of asymmetry seems to be correlated in some
way with the degree of differentiation or development, so that the
greater the degree of development the more stable is the asymme-
try of the organism.

SUMMARY

1 Inthe first four stages of the lobster’s development, a crusher
may be produced on either the right or the left side of the body by
the autotomous amputation of the chela on the opposite side—the
regenerated chela becoming a nipper.

2 During the fifth stage, although the chele are apparently
still symmetrical, the possibility for such experimental control
disappears.

Control of Asymmetry 483

3 In later stages of development, when the asymmetry of the
chele has become established, the amputation of one or both
chelze does not produce a reversal of the original asymmetry.

4 The results of these experiments indicate therefore that
the factors which control the asymmetry of the lobster become
operative after hatching and are correlated with conditions of
growth after the organism leaves the egg. No indication was
found that the occurrence of right or left relations of asymmetry
in this species can be referred to germinal units having “alter-
nating dominance.” It appears also that these relations are not
due to an “inverse organization of the egg,”’ for it is evident that up
to the fifth stage right or left asymmetry can be produced at the
will of the experimenter.

January, 1908

LIST OF LITERATURE

Conxuin, E. G. ’03—The Cause of Inverse Symmetry. Anat. Anzeiger, Bd. xxiii,
1903, p. 577-588.

’05—The Mutation Theory from the Standpoint of Cytology. Science,
n. s., vol. xxi, 1905, p. 525-529.

Emmet, V. E. ’05—The Regeneration of Lost Parts in the Lobster. 35th Report
of Inland Fisheries of Rhode Island, 1905, p. 81-117, with two
plates.

’06'—The Relation of Regeneration to the Moulting Process of the Lob-
ster. 36th Report of Inland Fisheries of Rhode Island, 1906, p.
257-313, with four plates.

’06*—Torsion and Other Transitional Phenomena in the Regeneration of
the Cheliped of the Lobster. Journ. Exp. Zodl., vol. ili, 1906, p. 603-
618, with two plates.

’06'—The Regeneration of Two Crusher Claws following the Amputation
of the Normal Asymmetrical Chele of the Lobster. Archiv f.
Entwick-Mech., Bd. xxii, 1906, p. 542-552, with two plates.

’o7'—Regeneration and the Question of ‘Symmetry in the Big Claws of
the Lobster.’ Science, vol. xxvi, 1907, p. 83-87.

‘o7*—Regenerated and Abnormal Appendages in the Lobster. 37th
Report of Inland Fisheries of Rhode Island, 1907, p. 99-152, with

ten plates.

484 Victor E. Emmel

Hap ey, P. B. ’05—Changes in Form and Color in Successive Stages of the Amer-
ican Lobster. 35th Report of Inland Fisheries of Rhode Island, 1905,
p- 44-80, with eleven plates.
Herrick, F. H. ’95—The American Lobster. Bull. U. S. Fish Commission., 1895,
252 pp., with 54 plates.
’o7—Symmetry in the Big Claws of the Lobster. Science, vol. xxv,
1907, P- 275-277-
Morean, T. H. ’04—Notes on Regeneration. Biol. Bull., 1904, vol. vi, p. 159-172,
with four figures.
’°o7—Experimental Zodlogy, New York, 1907, 454 pp.
PrzipraM, H. ’o1—Experimentelle Studien tiber Regeneration. I. Arch. f. Entw.-
Mech. Bd. xi, p. 321-345, with four plates.
*o2—Experimentelle Studien uber Regeneration. II. Arch. f. Entw.-
Mech., Bd. xiii, p. 507-527, with two plates.
’07—Die Heterochelie bei decapoden Crustaceen. III. Arch. f. Entw.
Mech., Bd. xix, p. 181-247, with six plates.
’o7—Die Scherenumkehr bei decapoden Crustaceen. Arch. f. Entw.
Mech., Bd. xxv, p. 266-345, with five plates.
Witson, E. B. ’03—Notes on the Reversal of Asymmetry in the Regeneration of
the Chelz in Alpheus heterochelis. Biol. Bull., vol. iv, p. 197-
214, with three figures.
ZeELENY, C. ’05—Compensatory Regulation. Journ. Exp. Zodl., vol. ii, p. 1-102,
with twenty-nine figures.

THE PHYSIOLOGICAL BASIS OF RESTITUTION OF
EOsSt,PARDS

C. M. CHILD

Hull Zotlogical Laboratory, University of Chicago, Chicago, Ill.

With One Ficure

In a series of “Studies on Regulation” which have appeared
in Roux’s Archiv and the Journal of Experimental Zodlogy during
the last five years, and in certain other papers (Child ’o6a, ’06b),
I have attempted to point out the essentially functional character
of form-regulation and have defined regulation in general as a
return or approach to physiological equilibrium after such equi-
librium has been disturbed or altered (Child ’o6a). According to
this idea form-regulation and functional regulation are both essen-
tially the same thing. Itis perhaps unnecessary to state again here
what I have repeatedly stated, viz: that the term “functional”
is used in this connection in its widest sense as equivalent with
“dynamic” or “physiological” and so includes all dynamic fac-
tors in organic life. In other words, the problem of form-regulation
is a physiological problem and not a problem swt generis as Driesch
and various other authors have maintained.

Let us consider for a moment what these assertions imply as
regards the factors concerned in the determination of any par-
ticular structure. If we assert that a given structure is altered or
determined by functional conditions does not this assertion neces-
sarily involve the idea of relation to its environment, intra-organic
or extra-organic or both? As a matter of fact the very essence
of the term “functional” as employed in these papers is to be
found in the interrelation or correlation between the different parts
of the organism and between the.organism and its extra-organic
environment.

Tue Journat or EXPERIMENTAL ZOOLOGY, VOL. V, NO. 4

486 C. M. Child

That this could fail to be evident to any reader of these papers
had not occurred to me until a recent paper by Prof. S. J. Holmes
(Holmes ’07) came to my notice. ‘This paper is a restatement of
the author’s symbiotic theory of form-regulation and a reply to
certain criticisms of my own (Child ’o6a) of an earlier statement
of this theory (Holmes ’04). Holmes maintains that my sugges-
tions concerning the nature of form-regulation do “not contain
any general principle of explanation for that functional substitu-
tion and equilibration upon which it is assumed that form-regu-
lation depends. But I suspect that when his theory comes to be
developed so as to supply this missing element it will involve the
assumption of some such symbiotic relation between the parts of
the organism as I have assumed ” (p. 424). If I understand this
assertion, it involves a serious misapprehension of my position. I
have insisted again and again in my work on form-regulation in the
interrelations or correlations between parts—in fact, certain of my
papers have been concerned chiefly with showing that such rela-
tions existed. Moreover, it is in consequence of the existence of
such relations that I regard form-regulation as essentially a func-
tional process. Even in my earliest papers positive statements
on this point were made. ‘Thus, for example, on p. 219 of No 1
of my Studies on Regulation (Child ’o2) in a consideration of the
general body-form of Stenostoma I wrote: “Every organism is
what it is because of the relation of all its parts to each other and
to the rest of the world. If any of these relations are changed the
organism is changed.” And again in No. IV of the Studies
(Child ’o4a) in a discussion of “formative factors:”’ ‘ All the com-
plex activities of which organisms are capable are ‘formative
factors:’ when we can view all of these in their complex inter-
relations, then and then only shall we ‘understand’ organic form.”
Also in No. V (Child o4b): “The factors of organic form include
all the activities of organic substance as well as the environmental
factors in varying degree. Indeed, in most cases, if not in all, we
may regard organic form as the visible effect upon the protoplasm
of functional factors in the widest sense”’ (pp. 468-469). In the
later papers these interrelations are still more strongly emphasized.
I have preferred not to designate them as symbiotic relations since

Physiological Basts of Restitution of Lost Parts 487

I cannot see that anything is gained by the use of this term. More-
over, it seems to me that many of the correlations are not really
symbiotic at all, except in so far as they may be mutual with
respect to complex parts of the organism. For example, the mere
mechanical union with other parts is undoubtedly in many cases
one factor in preventing parts of the organism from undergoing
regulation into wholes. But I cannot see that it serves any useful
purpose to call such factors symbiotic relations. It seems pref-
erable therefore to maintain that these relations, or as I believe
we may more properly call them, correlations, are physically and
chemically of all sorts possible in the material and environment
in which they exist. Moreover, while many of them are undoubt-
edly mutual,i. e., reciprocal, at least as regards complex parts,
others are just as certainly largely or wholly one-sided so far as
form is concerned. It seems scarcely necessary to enlarge further
upon this point. As regards the existence of relations between
parts as an essential feature of regulation Holmes and I agree
perfectly. As regards form-regulation we differ, in that it seems
to me difficult or impossible to account for the facts on the basis
of symbiotic relations, even in the widest sense.

Holmes’ illustration of the process of regeneration is as follows:
“Let us imagine an organism made up of a number of differen-
tiated cells, each of which derives some advantage from some
substances produced by the contiguous cells, and giving out some
substance upon which the contiguous cells are more or less depend-
ent. We will suppose that in addition to these differentiated cells,
there are scattered through the body numerous indifferent or
embryonic cells whose multiplication is held in check by the others,
but which upon the removal of any part respond to the functional
disturbance by growth and multiplication near the place of muti-
lation. We may represent our organism by the following diagram
in which the differentiated cells are represented by the larger
circles 4, B, C, etc., and the indifferent cells by the smaller circles
between them. Each cell such as 4 contributes something util-
ized by B, G, and F, and derives something in return from each
of these sources. Now suppose 4 is removed: the indifferent
cell lying nearby, no longer held in check by the same stimuli,

488 C. M. Child

begins to grow and develop. What line of differentiation will it
most naturally take? Owing to the symbiotic relation existing
between the cells differentiation in the direction of 4 will be most
favored as this secures it the advantages which 4 received. In
other words, this will be the direction of development along which
social pressure will tend to guide it. And the result will be a regen-
eration of the missing part” (Holmes ’o4, p. 282; ’07, pp. 420,
411).

In 1906 (Child ’o6a) I criticised this illustration on the ground
that if the cells or parts were mutually dependent, 1. e., if sym-
biotic relations existed between them, removal of any one of them,
e. g., 4, would bring about changes in the others in consequence
of which their influence upon the undifferentiated cell substi-

Fic. 1

tuted for 4 would be different from what it was originally, and
hence the undifferentiated cell or part would develop—not into
another 4 but into something else. There is logically no escape
from this conclusion. ‘The removal of 4 results in the formation
of a new system different from the original and must necessarily
do so, except under certain limiting conditions to be discussed
below.

Holmes’ reply to my criticism is as follows: “According to
Child, since the removal of 4 would alter B, G, F, etc., not only
something different would be developed in place of 4, but the
whole complex, according to my theory, would be profoundly
altered. How far this tendency will result in a modification of
these cells depends on the plasticity of the organism and the degree

Physiological Basts of Restitution of Lost Parts 489

of mutual dependence of the parts—factors of course which vary
in different organisms. But Child overlooks the fact that accord-
ing to the symbiotic relation assumed, the other cells C, D, £, etc.,
tend to keep B, F, Gin their original condition. In so far as these
remain in their original state, their influence on the indifferent
tissue in the region of 4 will tend to mold it in the direction of the
missing parts. In so far as B, G and F are modified through the
loss of the missing part, their influence on the tissue in the region
of A will come to be modified, and they will in turn modify the
cells lying next to them. But, as there is a tendency for the modi-
fications produced by the loss of 4, to spread successively to other
parts, there is also a tendency, according to my theory, toward
the checking and reversal of this process. Ifthe loss of 4 tends to
modify B, F and G, the presence of £, C and D tends to hold them
in place, and in so far as these are maintained through this influence
they tend to mold the tissue in the position of 4 into the form of
the missing part; and in so far as this is so molded, its modifying
influence on B, F and G is diminished”’ (Holmes ’07, pp. 425,
426).

I am unable to see that this argument shows that something like
A may be generally replaced. Undoubtedly the modifying in-
fluence of 4 upon the contiguous cells or parts B, F, G, is lessened
by the presence of other cells or parts, E, C, D, but it is not reduced
to zero in any case where the relations between parts are mutual.
The balance between the “tendency for the modification produced
by the loss of A to spread” and the opposite tendency simply deter-
mines how great or how small the modification shall be. Some-
thing more or less like 4 may undoubtedly be produced in many
cases, but according to this hypothesis we should expect that the
regenerated part would differ more or less widely from the original
part in most cases.

In fact, if the restoration of a part removed is purely a matter of
interrelation between the various parts of the system, we must modify
this hypothesis in either one of two ways to account forit. First: we
may assume that the removal of the part, 4 in Holmes’ diagram,
does not alter the other parts, B, G, F, etc., in any way which
affects essentially their interrelations with the parts of the system.

4.90 C. M. Child

In this case the undifferentiated material which in the absence of
A is stimulated to develop, will develop into another 4. But in
this case the relation between the original 4 and the other parts
of the system is essentially one- iced and not mutual or sym-
biotic. Or as a second possibility, we may assume that the rela-
tion between 4 and the other parts of the system is such that
removal of A produces modifications in the other parts only very
slowly, while in the absence of 4 these other parts affect the undif-
ferentiated cells in such manner as to bring about rapid develop-
ment so that restoration is complete before the parts B, G, F, etc.,
have been appreciably altered by the absence of 4. Here the
relations, though in the final analysis mutual, are so far as 4 and
its restoration are concerned, one-sided. In short, if we accept
the symbiotic theory as a basis, we can account for the restoration
of a part like that removed only by additional assumptions, accord-
ing to which the relations inv olved in the restoration become prac-
tically one-sided rather than mutual.

In my earlier criticism of Holmes’ theory (Child ’o6a, pp. 420,
421) the following statement appears: “To return to Holmes’
diagram, replacement of 4 can occur only when the relation is
largely one-sided, i. e., when 4 is dependent on B--F, but these
latter are not to any marked degree dependent on A. In this case,
and in this case only, will the “‘social pressure” force the undif-
ferentiated cell to differentiate into something like 4.”’

Holmes replies to this: ‘Where redifferentiation from new
tissue is concerned, as in the present case, it is not the relation of
A to B-—F, that should be more or less one-sided, but the relation
of the tissue in place of 4 to this complex. This is an important
distinction which Child does not seem to have considered. B—F
are relatively fixed, the tissue in place of 4 is young and plastic,
and more dependent so far as the direction of its differentiation
is concerned, upon b-f, than these are upon it. We may grant
that when regeneration occurs, the relation of dependence between
the old parts and the new tissue is more or less one-sided, although
the relations of the part removed may not have been. This would
naturally result if the parts were relatively stable. They may be
in a symbiotic relation, nevertheless, each part contributing in

Physiological Basts of Restitution of Lost Parts 491

some way to the normal functioning of the others, and dependent
to the extent that the removal of one part may alter only to a cer-
tain degree the quality and quantity of the activity of the surround-
ing parts, without producing extensive modification of structure
or function” (Holmes ’07, pp. 426, 427).

The first part of this argument seems to me to obscure the real
point at issue. If the relation between 4 on the one hand and
B-F on the other is not at least largely one-sided, removal of 4
must alter B—F, and if, as Holmes assumes, the new tissue which
replaces 4 is more dependent on B—F than they on it, it becomes
still more difficult to understand how the new tissue can replace
A, for, so far as B-F are concerned, it does not at first take the
place of 4 functionally. In the last sentence quoted, Holmes
attempts to save his symbiotic theory after admitting that in regen-
eration the relation may be more or less one-sided, by suggesting
the existence of symbiotic relations which do not produce “‘exten-
sive modifications of structure or function’? when one part is
removed. It seems to me that such relations are negligible quan-
tities so far as form-regulation is concerned, for if removal of a
part of the complex does not produce extensive modifications of
structure or function in the parts remaining, we must certainly
conclude that the presence of this part is not essential for the main-
tenance of the characteristic structure and function in the other
parts. Evidently then this assumption does not relieve us from
the necessity of assuming that the remaining parts are, so far as
form and structure are concerned, practically independent of the
part removed, 1. e., that the relations involved in form-regulation
are largely one-sided in cases where restoration of the missing part
occurs. It makes no difference whether we regard the persist-
ence of B—F in essentially unchanged condition after the removal
of A as due to “relative stability” or to real independence of 4.
The fact remains that 4 can be restored only in case the other
parts do persist essentially unchanged during the period between
its removal and its restoration to a certain stage of development.
And it is just as certain that Holmes’ symbiotic hypothesis cannot
account for such persistence except by assuming the existence of
special conditions which modify the relations between parts so

492 C. M. Child

that they become essentially one-sided rather than mutual. If
the restoration of a part like that removed were the exception
rather than the rule, or even if it were less frequent, we might still
accept the hypothesis. But a hypothesis which can account for
the typical phenomena within its field, only with the aid of addi-
tional special assumptions, which in this case amount practically
to throwing over the hypothesis, can scarcely be regarded as satis-
factory.

The numerous cases already known where an animal is capable
of replacing a part repeatedly after successive removals seem to
me to furnish additional evidence against Holmes’ theory. Even
so important a part as the head may be replaced repeatedly in
many forms without appreciable change in character. It is
scarcely probable, to say the least, that the relations between the
head-region and other parts are one-sided in the sense that it is
dependent on them, while they are independent of it. And if the
relation is not one-sided in this sense, we should expect that
repeated removals of the head would bring about essential changes
in the other parts, even if the first removal did not. If such
changes in the other parts do occur to any marked extent, it is
difficult to understand how a new head like the old can be replaced
time after time as the result of “social pressure,”’ for such changes
in the old parts must alter the character of the “social pressure.”
Here then Holmes’ theory leads us into something closely approach-
ing a dilemma.

Holmes continues: “If the parts B-F were more plastic,
absence of 4 would naturally tend to cause greater changes in
them, especially if new tissues were not produced in place of 4,
which would come to assume some of the missing functions before
the modification extended very far. There would then be a pro-
gressive modification extending from the region of 4, which would
tend to become less the farther it extended, but eventually perhaps
affecting more or less the entire organism. Functional equilib-
rium would then be maintained by working over the organism
so that all the parts were adjusted to functioning on a smaller
scale. The different methods of regulation, through morphal-
laxis, regeneration and the various combinations of these proc-

Physiological Basts of Restitution of Lost Parts 493

esses are, I believe, interpretable according to the symbiotic
theory, and the relations of regeneration and morphallaxis to the
degree of specialization of the parts which Child has elaborated,
are, in fact, exactly what the theory would lead us to expect”
(Holmes ’07, p. 427).

Here Holmes fails absolutely, so far as I can see, to explain how
and why equilibrium will be maintained. Certainly the “ pro-
gressive modifications” resulting from the removal of 4 cannot
bring the system back to its original condition: they must lead
either to destruction of the system, or rather of the parts of it
which remain, or else to a new condition of equilibrium different
from the old. How does Holmes know that “functional equilib-
rium would then be maintained by working over the organism
so that all the parts were adjusted to functioning on a smaller
scale?” What factor in the parts remaining compensates for the
“progressive modifications” resulting from the loss of 4? Why
should there be any compensation? ‘To none of these questions
does Holmes’ hypothesis give any answer.

According to the symbiotic theory as Holmes has presented it,
the removal of a part is, at least in many cases, analogous to re-
moval of a quantity from one side of an equation without change in
the other. It is obvious that such procedure alters the value of
one side of the equation in all cases except where the quantity
removed is equal to zero.

In short I believe that Holmes’ theory of regulation overlooks
the most essentral feature in the process of replacement of a part
removed. ‘This feature is the qualitative functional totipotence
of the remaining parts after removal of the part in question. In
other words, a part which has been removed cannot be replaced
unless something remains after its removal which plays its part
functionally in some degree. According to Holmes’ theory its
place is taken by undifferentiated tissue, which is forced to develop
into something like the part removed by the influence exerted
upon it by other parts. But this undifferentiated tissue cannot
exert the same influence on other parts as was exerted by the
part removed. Moreover it is difficult to understand how undiffer-
entiated tissue whose differentiation is held in check by the other

494. C. M. Child

differentiated parts, could persist in a system such as Holmes
postulated. If it has no function in the system and is not in
symb.otic relation with other parts, why should it not disappear ?
If, on the other hand, symbiotic relations between it and other
parts exist, it should, according to Holmes, differentiate into some-
thing. It is evident therefore that something besides undiffer-
entiated tissue must take the place functionally of the part removed
if replacement is to occur.

We can most readily gain an idea of what this something is by
means of a concrete example. In Planaria and various other
triclads, where even small pieces are capable of replacing all parts,
we find that the reactions of such pieces, while differing in degree
from those of the original animal, do not differ essentially in kind
After removal of the head, for example, the piece reacts in much
the same manner as when the head was present, though more
slowly and with less energy. In Leptoplana, on the other hand,
where regeneration of a head does not occur after removal of the
ganglia, the piece without ganglia is at once and clearly distinguish-
able from the animal with ganglia by the character of its reactions.

In Planaria then, and in the other forms where replacement of
the head and ganglia are possible, the piece still retains in some
degree the functional characteristics of a head-region. In remov-
ing the head we have not removed the only region possessing such
characteristics, but only the region which possesses them in the
highest degree of any part of the animal. In Leptoplana removal
of the head and ganglia leaves no part which can supply function-
ally, even in slight degree, their place, and formation of a new
head is impossible.

We must conclude that the localization of visible structural
differentiation in an organism is not necessarily coextensive with the
localization of functional processes or conditions characteristic of
th's region, but may be limited to the region of greatest energy of
these processes or conditions. It is a well-recognized fact that the
so-called functional structure of bone, tendon, etc., represents only
the most frequent or most energetic functional conditions, and
there is every reason to believe that similar relations exist between
structure and function in many other cases. The case of Planaria

Physiological Basis of Restitution of Lost Parts 495

cited above is in fact a demonstration that functional processes
may be less sharply localized than the structures which represent
them. The anatomical structure known as the head in Planaria
is not the only region where “‘head-reactions”’ are possible, but
it does represent the region where they occur with greatest energy
and frequency in the normal animal. Admitting this, the question
arises as to why heads do not form all along the body in Planaria,
i. €., as to why structure should be thus more narrowly localized
than function. The answer is not far to seek. If two parts, one
of which is capable of reacting in a certain manner more rapidly
and with greater energy than the other are correlated, the reaction
to a given stimulus ail occur in the first part earlier and with
greater energy than in the second. ‘The fact that a reaction has
occurred in the first part must bring about changes in the system
in consequence of which the character of reaction in the second part
is altered. If structure is, as I believe, the visible expression of
functional or dynamic conditions, we cannot expect that the second
part, even though it possesses in some degree the same functional
capacities as the first should exhibit the same structure, for the
very fact of its correlation with the first part which possesses these
capacities in greater degree determines that the functional con-
ditions in it shall be different from those in the first part. In gen-
eral terms we may say that the region where a particular functional
complex occurs with greatest energy, frequency or rapidity domi-
nates so far as this particular complex is concerned all other parts
of the organism which possess the same capacity in less degree,
and modifies their activities to a greater or less extent. Conse-
quently the structure with which a particular functional complex
is associated in the normal animal may be much more narrowly
localized than the functional complex. In Planaria, for example,
the head-structure is limited to the anterior end of the animal,
while the functional capacities commonly regarded as charac-
teristic of the head exist at all levels of the body. These other
regions are capable of producing a head-structure, but only when
isolated from the original head.

It is evident then from this consideration that localization of visi-
ble structure is not necessarily an exact criterion of localization of

496 C. M. Child

functional capacity. In all cases where a difference in localiza-
tion exists, structure is more narrowly localized than functional
capacity. On this fact, which I believe to be of fundamental
importance for the problem of form, depends the ability of a part
to become a whole when isolated.

In order to bring out clearly the difference between Holmes’
hypothesis and my own, we may make use of Holmes’ diagram
(Fig. 1). According to my hypothesis, the various parts, 4, B,
C, D, etc., though perhaps visibly different as regards structure,
each possess the physiological properties of the others or of some
of the others in some degree. B, Gand F, for example, the parts
contiguous to 4, are capable in some degree of activities similar
to those characteristic of 4, but as long as 4, a region of greater
energy or frequency or rapidity as regards these particular activi-
ties is present the correlations arising from it obscure, inhibit or
modify the activities of B, G, F, so that they appear structurally
and functionally to be different from 4. But when J is removed,
the parts B, G, F become at once the dominating parts as regards
the A-activities and the correlations between them and other
parts become similar in kind, to those which previously existed
between 4 and the other parts, though probably different in degree.
In short B, G, F, or certain portions of them are substituted func-
tionally for 4 simply because in the absence of 4 their activities
must, by virtue of their constitution, be somewhat similar to those
of A. No entelechy or other peculiar principle is needed to guide
or determine this substitution. It occurs with the same certainty
as any other physical phenomenon in all cases where these parts
possess the functional capacities to which attention has been
called above. ‘ According to this hypothesis, the undifferentiated
cells postulated by Holmes are not only unnecessary, but could
not substitute for 4 if present, because the parts B, G, F are more
like A than are the undifferentiated cells and would therefore
dominate in the process of substitution.

The d-processes are undoubtedly in most cases, if not in all,
at first less energetic or less rapid or both, than they originally were
in A, and in consequence of this difference the system may regain
its original condition of equilibrium in either one of two ways. If

Physiological Basis of Restitution of Lost Parts 497

the other parts C, D, E are plastic, 1. e., if their activities are
readily and rapidly altered by altered conditions, they will be
affected by the decrease in 4-correlations following the removal
of A and will undergo more or less change in response to the
changed correlations, 1. e., regulation by what we ordinarily call
redifferentiation will occur. If, on the other hand the parts C,
D, E are relatively stable, i. e., not rapidly changed by altered
conditions, they and the correlations arising from them will remain
much the same as before the removal of 4. But the 4-processes
in B, G, F are out of proportion to these correlations and must be
quantitatively increased by them. In this case then equilibrium
is regained by functional hypertrophy of the portions of B,G, F,
which are the functional substitutes for 4. This is what we know
as regeneration in the stricter sense, 1. e., formation of new tissue
from the regions adjoining the cut surface and its visible differen-
tiation with increase in size into a part like that removed. In
most plants and in some animals regeneration occurs from regions
more or less distant from the cut surface, simply because these
regions are physiologically more like the missing part than is the
region at the cut surface.

As a matter of fact, since correlations in the system are at least
in large measure mutual, most if not all cases of restitution
are mixtures of redifferentiation and regeneration. Some change,
1. e., some redifferentiation occurs in some or in all parts of the
system and some regeneration, 1. e., functional hypertrophy of the
part which forms the physiological substitute for the part removed
takes place.

Holmes’ hypothesis fails to recognize the fundamental fact,
viz: that something must remain after the removal of a part, 4,
which can take its place functionally in the system in some degree.
Without this the only factors which can prevent progressive depar-
ture from the original condition are lack of plasticity in the parts
remaining or one-sided relations between parts. As a matter of
fact however plasticity is a conspicuous feature in many forms in
which the regulation of parts into wholes occurs most readily,
and on the other hand all the evidence indicates that correlations
are in large measure mutual rather than one-sided. In those cases

498 C. M. Child

where a part after isolation is incapable of becoming a whole,
while the remaining parts are capable of replacing it, there is
reason for believing that the correlations are more or less one-
sided, i. e., the part in question has been so greatly modified by
the past or present correlations arising from other parts that it has
lost its totipotence and can never become a whole, but the correla-
tions arising from this part have not been sufficient to modify the
other parts of the system to an equal extent. Examples under
this head are the appendages of arthropods, amphibia, etc.

One other point discussed by Holmes requires brief considera-
tion: in his first paper he selected the regulatory development of a
head in Planaria as an illustration of the working of social pres-
sure. In his discussion of this case differentiation is regarded as
proceeding from the cut surface distally, in consequence of the
social pressure exerted on the new parts by the old (Holmes ’o4,
pp. 282, et seq.). In my criticism of this point (Child ’o6a, p.
421, et seq.), I called attention to the fact that in Planaria, and in
other forms as well, differentiation of the regenerating tissue actu-
ally proceeds in the opposite direction, 1. e., from the tip toward
the base. In reply to my criticism Holmes (’07, pp. 427, 428)
points out that the first visible differentiation is not necessarily
the first actual differentiation, that “before any external features
are produced in the development of a limb the main outlines of
its differentiation may have been established through influence
proceeding from its basal part, after which the tip might differen-
tiate more rapidly than the intervening portion and the other visi-
ble features of structure appear successively toward the base.”
He also points out that in many cases the visible differentiation
is centrifugal rather than centripetal and cites the case recently
described by Zeleny (’07) of the antennule of Mancasellus, in
which visible differentiation at first proceeds from the base
toward the tip, but later in the opposite direction. He continues:
“But granting that, in many cases, differentiation actually begins
at the extremity and works toward the base of the regenerating
organ, the process is not inconsistent with the point of view here
set forth. We may suppose that the influence of the environ-
ment causes the extremity of an organ to begin to differentiate

Physiological Basis of Restitution of Lost Parts 499

like that of the missing part. That is only one step. We have
then to account for the numerous coordinated differentiations
that take place as the part develops toward the base. * * * The
fact that, with few exceptions, such as the failure to regenerate the
intermediate segments of the appendages, etc., the whole organ,
nothing more nor less, is regenerated, and forms a congruent union
with the basal part, is indicative of close interaction of the various
parts of developing organs with the body of the organism at all
stages of the process.

“T am inclined to think that neither centrifugal nor centripetal
differentiation, expresses the entire truth of the matter, but that
the new part differentiates as a whole, much as organs doin embry-
onic development, and at all times in intimate fictional relations
with the old part, differentiation becoming accelerated in one part
or another, according to special conditions’’ (Holmes ’07, pp.
428, 429).

As regards most of these points my position does not differ very
widely from that of Holmes. My criticism of his analysis of the
case of Planaria was directed primarily, not at his hypothesis in
general but merely at his failure to consider the actual facts in
that case. [see no reason why the occurrence of differentiation
in either direction or in both should constitute a fatal objection
to his hypothesis or to my own, for such differences are merely
incidental and depend on the conditions in individual cases.
When my criticism was written the experimental data seemed to
indicate that visible differentiation in the centripetal direction
was the general rule, though by no means without exceptions, and
since Holmes did not in his first paper attempt to account for
this fact in any way, his hypothesis was open to criticism. I cer-
tainly had no intention of maintaining that differentiation must
in all cases proceed centripetally, since at that time various cases
were known to me in which visible differentiation proceeded centri-
fugally.t. Ido not believe however that Holmes’ suggestion that

1In his discussion of the direction of differentiation in the antennule of Mancasellus, Zeleny (’07, p
335) says: ‘Child has recently expressed the opinion that differentiation must in every case proceed
from the tip toward the base and in no other way.”’ My actual statement was that differentiation from

the tip toward the base is ‘‘a general ruel in cases of regeneration.” ‘This statement as it stands is

500 C. M. Child

the new part differentiates as a whole, much as organs do in
embryonic development is universally applicable. There are
certainly many cases in which the terminal portions attain or
approach their final condition of functional activity before the
basal parts are formed, and in a considerable number of cases
also the basal parts are replaced incompletely or not at all. In
fact it seems to me that such cases might be expected to occur,
for in a correlated system the conditions for the regulatory forma-
tion of non-terminal regions must, at least sometimes, be largely
dependent on the existence of typical functional conditions in
terminal parts. If conditions in the terminal parts are more im-
portant than those in the old parts as determining factors in the
differentiation of intermediate parts we should expect to find the
intermediate parts differentiating later than the terminal parts,
but if, on the other hand, conditions in the old parts are the chief
determining factors, differentiation might occur wholly in the
centrifugal direction.

Moreover, although I agree with Holmes that the absence of
visible differentiation does not necessarily imply absence of physi-
ological differentiation, I am incined to believe that the direction
of progression of visible differentiation is not without significance
as an indication of the direction of progression of physiological
differentiation. In other words, while the absence of visible
differentiation proves little or nothing with regard to physiological
differentiation, its presence may prove something. I think it
probable therefore that in some cases the regenerating part is not
differentiated as a whole, but that its various regions are deter-
mined successively in one direction or theother: in other cases
it may perhaps be differentiated as a whole. It would appear
that none of these possibilities conflict with either Holmes’
hypothesis or my own.

undoubtedly open to misinterpretation and should have been qualified, for I was well aware at the time
it was made that centrifugal differentiation occurred in various cases. In fact, I had shown in earlier
papers (e. g., Child, ’o4b) that the differentiation of the intestine in regenerating parts of Leptoplana
is apparently centrifugal. However I take the present opportunity to make acknowledgments to
Holmes and Zeleny for calling my attention to this misleading statement, and also to make clear my
teal position in the matter, which is that differentiation may occur in either direction or in both accord-

ing to conditions in the particular case.

Physiological Basis of Restitution of Lost Parts 501

To sum up: Holmes and I agree in that we both postulate a
condition of physiological equilibrium, or rather, as I should put
it a condition of oscillation or cyclical change about equilibrium,
asthe basisof ourhypotheses. The chief pointof difference between
us is that Holmes’ hypothesis does not, as | understand it, provide
for the maintenance of or return to the typical condition, except by
the assumption of relations largely one-sided, or that of lack of
plasticity. While these assumptions may serve for certain indivi-
dual cases, they seem to me to be totally inadequate for the analysis
of form-regulation in general. According to my own hypothesis
a part can be replaced only when some other part is physiologi-
cally sufficiently similar to it to perform its functions at least
qualitatively, if not quantitatively, after its removal.

The independent formulation of two hypotheses of form-regu-
lation so similar in general point of view as are Holmes’ and my
own, is I believe not without significance, since agreement between
different observers as regards the general nature of problems may
be an indication that real progress inthe analysis of data is being
made. It is desirable in such cases, and particularly in fields
where the data are so varied and complex, that differences of
opinion should be fully and critically discussed. For this reason
I have ventured to consider at some length in the present paper
the points which seem to me debatable, and to state my own posi-
tion in a manner which I hope will lessen the chances of future
misunderstanding.

Hull Zodlogical Laboratory

University of Chicago
February, 1908

502 C. M. Child

BIBLIOGRAPHY

Cuitp, C. M. ’02—Studies on Regulation. I. Fission and Regulation in Stenos~
toma. Arch. f. Entwickelungsmech., Bd. xv, H. 2 and 3, 1902.
‘oga—Studies on Regulation. IV. Some Experimental Modifications
of Form-Regulation in Leptoplana. Journ. Exp. Zo6l., vol. 1, no. 1,
1904.
‘ogb—Studies on Regulation. V. The Relation between the Central
Nervous System and Regeneration in Leptoplana: Posterior Regen-
eration. Journ. Exp. Zodl., vol. 1, no. 3, 1904.
‘o6a—Contributions toward a Theory of Regulation. I. The Signifi-
cance of the Different Methods of Regulation in Turbellaria. Arch.
f. Entwickelungsmech, Bd. xx, H. 3, 1906.
‘o6b—The Relation between Functional Regulation and Form-Regula-
tion. Journ. Exp. Zodl., vol. in, no. 4, 1906.
Houmes, S. J. ’04—The Problem of Form-Regulation. Arch. f. Entwickelungs-
mech. Bd. xvi, H. 2 and 3, 1904.
*o7—Regeneration as Functional Adjustment. Journ. Exp. Zodl., vol.
iv, no. 3, 1907.
ZeLeny, C. ’07—The Direction of Differentiation in Development. I. The
Antennule of Mancasellus macrourus. Arch. f. Entwickelungs-

mech, Bd. xxii, H.2, 1907.

THE PROCESS OF HEREDITY AS EXHIBITED BY THE
DEVELOPMENT OF FUNDULUS HYBRIDS!

BY
H. H. NEWMAN

With Five Pirates AND SixTEEN Ficures 1N THE Text

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' Contributions from the Zoological Laboratory, University of Michigan (No. 116), and from the
V oods Hole Marine Biological Laboratory. :

Tue JourNar or ExrperiMENTAL ZOOLOGY, VOL. V.NO. 4

504 Elewile Newman

I. INTRODUCTION

The experimental work on heredity of the last decade or so
has, I believe, dealt too exclusively with definitive characters and
has overlooked the origin and development of these characters.
The usual method of procedure in such experiments 1s to inter-
breed two species or varieties exhibiting well marked differenti-
ating characters, usually of a superficial sort, with the idea of deter-
mining for each character of the one species or variety whether it
blends with or dominates the corresponding character of the other
species or variety. The characters studied are nearly always
definitive characters and can be observed only in advanced young
or in adults.

Such work, it seems to me, overemphasizes the importance of
one stage in the developmental process. That the definitive
stage of a character is a comparatively fixed one does not obviate
the necessity of studying the origin and ontogeny of such a charac-
ter. But in the kind of work referred to above all other stages
in the process, no matter how interesting and instructive, are
overlooked. ‘This tendency to regard the condition of characters
in the adult as the whole of heredity has led to the publication of
this paper as a plea for the study of heredity as a developmental
process. In the present work it appears that sometimes the ma-
ternal and sometimes the paternal influence is uppermost. An
individual or a strain may in early stages show a predominance of
maternal characters and later a predominance of paternal, while
in between the first and last stages may be seen an apparent struggle
between the paternal and maternal forces, expressing itself in an
alternating dominance of one or the other.

The work consists of observations of the comparative devel-
opmental histories of two pure breeds and their reciprocal
crosses, the study being continued from fertilization until long
‘after hatching. It makes no pretense of being a thorough-going
study of Teleost embryology. Only such points in the devel-
opmental processes are noted as proved themselves efhcient for
the comparative study of rates of development, sizes of whole
or of parts, degree and kind of pigmentation, functional activity,

The Process of Heredity 595

physiological resistances, etc. Only to this extent then is the
developmental history of the bony fish treated, hence no apology
is offered for the incompleteness of embryological detail.

The experiments of Boveri, Driesch, Herbst, Seeliger, Fischel,
etc., have for this work only a casual significance for the reason that
the prime interest of the authors was focused upon another prob-
lem, viz: the comparative potency of nucleus and cytoplasm in
heredity. No attention was paid to heredity as a process and
this is the chief idea brought out in the present work. No formal
review of this rather voluminous literature on Echinoderm hybrids
is attempted in this place. Only where the facts presented have
a distinct bearing on the work in hand will the data of these
authors be referred to.?

Probably no type of egg offers so many advantages for the
study of heredity as a process as that of the bony fish, and it is
equally probable that few species of fish are so available for
this kind of work as those used in the breeding experiments here
described. Some of the most obvious advantages of the species
used are as follows:

1 Both species are abundant and easily obtained.

2 Both species thrive well under laboratory conditions.

3 The adults are of convenient size, neither too large nor too
small, and yield large numbers of eggs that are easily stripped.

4 The spawning behavior and sexual dimorphism of both
species were easily studied and a knowledge of these phenomena
proved to be a prerequisite for the present study.

5 The adults possess a sufficiently large number of differenti-
ating characters to furnish data for the comparative study of adult
hybrids, should it prove feasible to rear the young fish to maturity.

6 The eggs of both species are of convenient size, and, what is
of more importance, are of quite unequal size.

7 ‘The eggs are nearly transparent and the development of all
characters can be studied without difficulty in living material.

8 ‘The embryos can be studied for long periods as stationary

“For a comprehensive review of recent work on Echinoderm hybrids, see Alfred Fischel’s paper,

“Ueber Bastardierungsversuche bei Echinodermen.” Arch. f. Entw.-Mech, 22, 1906.

506 J&L. Ele Newman

objects within an envelope, needing no especial care and provided
with their own food.

g The egg membrane of both species is a thick, resistant
capsule, capable of protecting the developing embryos from the
various detrimental factors of their environment and making it
more nearly possible to study heredity uninfluenced by extraneous
phenomena.

10 Many other special advantages that apply only to the two
species used will receive notice in the body of the paper.

II MATERIAL AND METHODS

Materials—A Descriptive Account of the Species Used

Fundulus majalis and Fundulus heteroclitus, two species of
killifish belonging to the family Poeciliide, furnished the material
for the bre dine experiments detailed below. Both species abound
along the Atlantic coast and are familiar to all workers at the
Woods Hole Marine Biological Laboratories, where this work
was done.

These two species interbreed readily and one of the reciprocal
crosses is capable of hatching and of living in aquaria at least
seven months after hatching. The two species present well
marked differentiating characters in adults, embryos and eggs,
both morphologically and physiologically. An account of these
differences will furnish a logical introduction to the body of the
work.

1. Morphological Differences Between the Adults

A reference to Plate I will serve to show the strikingly different
general appearance of the two species. The actual average size
of mature adults is represented in the figures and it will be readily
seen that F. mayjalis is the larger though more slender species. A
pronounced sexual dimorphism is exhibited in both species.
This phenomenon is concerned chiefly with the shape and size
of the fins, color pattern, degree of pigmentation, the presence in the
males during the breeding season of minute “contact organs” that
are of use in clasping the female, etc. Although this sexual

The Process of Heredity 507

dimorphism would seem to be of no moment for the purposes of
this paper, since I have not as yet been able to rear hybrids to
maturity, yet a knowledge of this phenomenon is necessary as an
aid to understanding the methods employed. Under a subse-
quent heading is given a brief treatment of the essential points
about spawning behavior and sexual dimorphism.

2 Physiological Differences Between the Adults

a_F. heteroclitus is markedly more resistant to adverse con-
ditions, such as foul water, lack of oxygen, presence of carbon
dioxide, etc., than is F. majalis.

b F. heteroclitus is therefore found in habitats unfit for F.
majalis, such as brackish and foul ponds, etc.

cF. heteroclitus is much less readily affected by confinement
in small aquaria and carries on its spawning in captivity with
perfect freedom. F. mayjalis, on the other hand, is rather sullen
in confinement and frequently refuses food for some time after
capture. Spawning in aquaria is very rare with F. majalis.

d ‘Yhe flesh is harder and the muscles stronger in F. majalis
than in F. heteroclitus.

e The heart-beat, and hence the general circulation, is markedly
less rapid in F. heteroclitus than in F. majalis.

These and other differences between the adults that might be
mentioned concern us much less directly than do differences
between the eggs and developing embryos of the two species.

3. Morphological Differences Between the Eggs and Embryos

a The eggs of F. majalis are decidedly larger than those of
F. heteroclitus, the average diameter of the former being 2.7 mm.
and that of the latter 2 mm. A calculation shows that the vol-
ume of the average F. mayjalis egg is over twice that of the average
F. heteroclitus egg.

b The eggs of F. mayalis are of a decidedly yellowish color,
while those of F. heteroclitus are almost colorless and more
nearly transparent. “These differences are due to a different com-
position of yolk and protoplasmic content.

508 H. H. Newman

c The capsule surrounding the egg of F. heteroclitus is,
after exposure to water for some time, decidedly fibrous and
sticky, causing the eggs to clump up in a very disagreeable
fashion. The capsule of the F. majalis egg is, on the other
hand, scarcely fibrous or sticky and the eggs seldom clump. On
this account they are more easily handled than those of the other
species.

d The size of the developing embryos and of young fish on
hatching is in the two species in proportion to the comparative
volume of the eggs, that of F. majalis being about twice that of
F. heteroclitus.

e The color pattern of the young fish before and after hatching
is quite different in the two species, as are also the size and struc-
ture and pigment content of the chromatophores.

4. Physiological Differences between the Eggs and the
Developing Embryos

a The eggs and developing embryos of F. majalis, like the
adults, are much less resistant to unfavorable environmental
conditions than are those of F. heteroclitus.

b The eggs of F. heteroclitus reach the hatching period in
about two oe on the average, while those of F. mayjalis require
nearly three weeks on the average. As a corollary to this F’. het-
eroclitus is at all stages of development markedly in advance of
F. majalis. The two species on hatching are at same stage of
development.

c The body and yolk of F. heteroclitus embryos become
heavily pigmented after about three days of growth, while in
F. majalis only a very faint pigmentation occurs until after seven
or eight days. The color on the bodies of newly hatched young
is much paler in F. majalis than.in F. heteroclitus.

d The heart-beat of F. majalis is, stage for stage, much more
rapid than that of F. heteroclitus.

e A fair percentage of hybrids from F. heteroclitus eggs hatch
spontaneously and are capable of living and thriving for months
while none of the hybrids from F. majalis eggs ever hatch.

The Process of Heredity 509

A number of other differentiating characters might be listed
here, but it seems advisable to defer mention of many such charac-
ters until they can be treated in a connection more intelligible.

Method

Since the problem in hand has proven to be so largely one of
method, it seems necessary to preface the bare statement of the
method finally evolved with an historical account of some of the
steps in the evolution of this method.

The first season’s work brought out so much of contradiction
and ill success in rearing both pure and hybrid strains, that it
seemed necessary to become more familiar with the physiology
and behavior of the two species of fish used. ‘This study was
carried on during the early part of the second season and resulted
in the discovery of many interesting facts about the spawning
behavior, the significance of the sexual dimorphism displayed,
and the sure signs, morphological and physiological, of high sexual
tone. Although a full account of these phenomena has been
published,? it will be convenient in this place to set down some of
the facts that have a bearing on the present work.

1 Spawning Behavior and Sexual Dimorphism

These two species of fish, like other fish, have a well-defined
breeding season. That of F. majalis is somewhat earlier than
that of F. heteroclitus and lasts for a shorter time. In both species
there is a still more restricted period during which spawning is
carried on most actively, and during which both sexes are at the
height of their sexual tone. This sexual climax comes earlier
in F. mayjalis than in F. heteroclitus, by about two weeks, and over-
laps the corresponding period of the latter by about three or four
weeks.

During this period of a few weeks, which may be called the
spawning period proper, both species show marked changes in
structure and behavior. In the males an intensification OF pig-
ment appears over the entire body and especially in certain

3H. H. Newman. Biol. Bull., vol. xii, no. 5, April, 1907.

510 H. H. Newman

regions. A sort of steely glint, somewhat akin to iridescence
suffuses the body. On certain well-defined regions of the body
appear many characteristic, somewhat stiff, papilla that are readily
visible to the naked eye. These papilla are temporary organs
of definite structure, that appear only during the spawning season
and disappear afterward. They occur on dorsal and anal fins,
on the cheeks, and on the sides, regions that come into most
intimate contact with the body of the female during the act of
spawning. These papilla I have chosen to call “contact organs.”

In the female the body becomes paler than usual, the flesh
becomes soft, the fins soft and pliable, and the abdomen dis-
tended with eggs. They also show a coyness of behavior that
seems to incite the males.

The spawning act proper, as observed both in aquaria and
in the open, is essentially a clasping phenomenon. The male
pursues and corners the female, crowds his body against hers and
clasps her just anterior to the tail with his large, strong, and
especially modified dorsal and anal fins. While united in this
fashion, both bodies bent laterally into the shape of a flattened S,
the tail regions vibrate rapidly for from one to three seconds and
eggs and milt are simultaneously extruded in close proximity to
one another, thus insuring fertilization. An account of courtship,
rivalry and display, previous to spawning, while of some interest,
would take us rather far afeld at present. In a former paper
these subjects are fully treated.

This study of spawning behavior and the physical signs of
high sexual tone explained some of the earlier contradictions
and ill success and served greatly to improve the methods used.

2 The Importance of Equalizing the Physiological Condition
of the Parents

Experience showed what ordinary judgment should have sug-
gested that,if one wishes to eliminate the factors of over-and
under-ripeness, staleness, ¢tc., of sexual products, it is necessary
to cross-fertilize only during the period when both species are at
their sexual prime. Only if this precaution be observed can one
obtain in different experiments even approximately uniform results.

T he Process of Heredity 5 Il

Not only must one be careful as to the time for starting experi-
ments, but individuals must be carefully chosen. Males and
females, full grown and sexually mature, as indicated by the sure
signs of the sexual climax mentioned above, should always be used,
for if eggs are stripped from females that are under-ripe only a
small per cent of the eggs are capable of fertilization and the devel-
opment of those is apt to stop short of completion. If males
that have either failed to reach or have passed the sexual climax
are used, their milt may be either entirely or largely ineffective
in initiating normal development.

The eggs and milt of fish kept longer than three or four days in
aquaria very frequently become Seles and very unsatisfactory
results have been obtained from their use. These stale eggs are
frequently capable of fertilization and of partial development, but
the embryos usually die before hatching. This precaution applies
especially to F. majalis, which very eeldom spawns in captivity
and hence females may carry eggs in the oviducts for weeks after
they would normally be extruded. F. heteroclitus females, on
the other hand, have a habit of ridding themselves of over-ripe
eggs without the assistance of the males. Consequently stale
eggs are seldom found in that species. Stale milt may be avoided
in both species by selecting only the most highly colored males,
with well developed contact organs. F. majalis males will not
retain their dusky spaw ning donation for more than a few days
In captivity, so there is fle danger of obtaining stale milt from
that source if reasonable care is alot

A multiplicity of parentage in a single batch of eggs must be
avoided, as there is considerable variability in the eggs taken from
different females and in the milt of different males according to
their size, age, and degree of sexual maturity. Unless the eggs
of one female are used for both pure and hybrid strains, and the
milt of one male for both species of eggs in one series of experi-
ments, an undue amount of variability ensues as the result of
extraneous factors that needlessly complicate the issue and frus-
trate all attempts to study pure heredity.

512 H. H. Newman

3. The Importance of Equalizing the External Conditions of the
Developing Embryos

After some experience it was found necessary to limit the num-
ber of eggs in one vessel and to keep these well separated. The
tendency to clump up, exhibited by the eggs of F. heteroclitus,
causes marked variations in time rate of development and _ulti-
mate success in hatching. If eggs are too numerous or too crowded
in one vessel only a few succeed in hatching and many abnormal
conditions are manifested. F. majalis embryos do not endure
crowding so well as do those of F. heteroclitus, so, as a rule, only
about half as many of the eggs of the former species are used in
an experiment as those of the latter species.

Several methods were used for keeping the vessels well aérated.
Running sea-water was used, but it was found that a rusty deposit
collected on the surface of the eggs. This deposit seemed to
interfere with development; probably interrupted the gaseous
respiratory exchange. Aquaria containing Ulva were used in
some of the experiments, but these did not prove very satisfactory
on account of the fact that the eggs lay still too long and frequently
became covered with Saprolegnia and other molds. The method
that gave the best results was simple. Large, flat, covered bac-
teria dishes were used and the water was changed daily by
pouring off the surface and filling up with fresh sea-water. In
this way disease was kept at a minimum and there was always an
abundance of well aérated water.

It is absolutely necessary to pick out all eggs that have not been
fertilized as well as embryos that die from time to time, as these,
if left in the vessels, will infect the healthy individuals.

4 The Attitude that Must be Taken Toward Variability

Another lesson taught by experience in dealing with large
numbers of eggs and embryos is, that the high degree of variability
in different strains and in different individuals of the same strain,
must not be regarded as an insurmoutable difficulty in an endea-
vor to arrive at definite results. Variability, here as elsewhere,
is to be expected, and one must accept it as he finds .it and must

The Process of Heredity 513

learn to see the outlines of fixity and regularity through the haze of
a confusing diversity of conditions.

No two strains develop at the same rate, but are retarded or
accelerated by various conditions of temperature, oxygen content
of the water, etc.; only when a pure and a hybrid strain are started
simultaneously and are treated alike, can any basis of comparison
be attained. :

Even in pure strains, moreover, there is a considerable amount
of variability; imperfect eggs are always present and give imper-
fect and unhealthy embryos. The great majority of eggs in pure
strains, however, especially in the case of F. heteroclitus, develop
at a practically uniform rate for the first week. There is always
a considerable degree of variability in respect to the hatching
period, some individuals hatching as much as four days later than
others.

In hybrids of both kinds the factor of variability is a much more
important one than in the case of the pure strains. Here the
range of variability is enormously increased and it is a matter of
considerable difficulty, especially in advanced stages, to decide on
the average or representative condition when a hundred or more
embryos are examined. Considerable practice, however, has
made it possible, with some degree of personal satisfaction, to
select a specimen that represents either the most prevalent condition
or a judicious mean between extreme variants. In some cases,
especially during the first four days of development, the selection of
a representative condition offers no serious difficulties. It must be
understood, then, that, in the description and figuring of such
representative individuals in the succeeding pages, these have been
arbitrarily selected by the writer and that his best judgment has
been used inthe selection. Where actual measurements or numer-
ical determinations of structures or functional activities could be
employed there was no difficulty in selecting the most prevalent or
the average condition.

A fuller discussion of this factor of variability, especially as it
applies to hybrids, follows the presentation of data.

514 H. H. Newman

5 Methods Proper

The methods of experiment and study were naturally the direct
outcome of the experience outlined under the three previous heads.
By taking the precaution to equalize, as far as possible, the physio-
logical condition of the parents and the external conditions of the
developing embryos, and, at the same time, allowing for the factor of
variability, it was possible to get results of a somewhat regular
and invariable nature.

The method of procedure that gave the best results was as
follows: Fresh, egg-laden females of good size of both species
were selected. The eggs of a F. heteroclitus female were then
stripped into one finger-bowl, those of a F. majalis female into
another. The eggs were then stirred up with the finger so that
those first extruded and those last extruded might be evenly
distributed. “Then about half of the eggs in the two bowls were
transferred to two other bowls. ‘Two males, one of each species,
at the height of their sexual tone, as indicated by their dark colors,
the presence of contact organs, etc., were then chosen. The milt
of the F. majalis male was stripped into a very little sea-water,
stirred and poured partly on one lot of F. heteroclitus eggs and
partly upon one lot of F. majalis eggs, being stirred up with the
eggs in both cases. After washing the hands in fresh water,
which certainly killed all adhering sperm from the F. majalis
male, the F. heteroclitus male was used to fertilize the remaining
two lots of eggs. After allowing the eggs in all four bowls to
stand for about fifteen minutes with the small amount of water
used in the fertilization process, the excess sperm was washed out
with fresh sea-water and the eggs were transfered to large, cov-
ered bacteria dishes, containing about a liter of fresh sea-water.
Usually from one to two hundred eggs were allowed to develop in
each dish. The water in these dishes was partially drawn off
and renewed nearly every day. Eggs were dissected apart when-
ever a tendency to clump up manifested itself. All dead eggs or
embryos were removed as soon as noticed. The water always
smelled sweet and fresh in cultures treated as described, and a
very large percentages of embryos developed and hatched in the
two pure strains, especially in those of F. heteroclitus.

The Process of Heredity 515

For purposes of study lots of about fifty eggs were drawn off
periodically with a large pipette into Syracuse watch glasses and
examined with the low powers of the compound microscope. ‘The
methods of study and observation were many and involved the
use of both living and preserved material. Comparisons of
differences between the four strains of each series were made in
various ways. In early stages actual counts of blastomeres were
made, both in living and in fixed material. In somewhat later
stages, when the blastodisc begins to spread out over the yolk,
diameter measurements of large numbers of blastodiscs were made
with the aid of the ocular micrometer. ‘The latter instrument was
used for measuring head and body diameters, and other dimensions
of more advanced embryos.

Camera lucida drawings of living individuals, selected as good
representatives of a strain, were made at selected intervals. These
camera drawings were made at the level of the table and showed
a magnification of 28 diameters. All drawings were made at
the same magnification. Details in some of the drawings were
filled in from fixed and stained material, put up at the time when
the drawings were made.

The study of fixed material was of great assistance in confirm-
ing the observations made upon living material, and frequently
added much new data.

Since no two series gave the same results, I have decided to
present the data of one successful series in the form of an abbrevi-
ated pictorial table (Plates I, II] and IV). Other series can be
briefly compared or contrasted with this. Any one of five or six
series might equally well have been chosen, but this one is fairly
typical and shows perhaps a little more than any of the other
series examined. Some facts, however, are better brought out by
other series. No one series is complete for the reason that one
can make observations only at intervals and important stages
may be passed over between observations. ‘The entire history,
however, can readily be pieced together from a considerable num-
ber of series.

The four strains of the series selected are figured in tabular
form, nine stages being figured during the first week of develop-

516 /Gb, Jak Newman

ment. It is difficult to study whole embryos within the egg mem-
brane for a longer period, as they become too opaque and the
fluids within the membrane are apt to become cloudy and to
render outlines too vague to admit of camera drawings being
made. It was necessary for purposes of drawing and observation
to dissect out from the egg membranes both hybrid and pure
bred F. majalis embryos.

If the specimens selected for drawing were active it was neces-
sary to quiet them with choloretone before drawings could be
made. It was frequently difficult to find a specimen that lay in
a position suitable for drawing and much patience was required
in order to find individuals that were at once typical and fortunately
disposed for drawing.

For convenience in reference the four strains pictured in the
table (Plates II to IV) are lettered H, h, m, and M, capital letters
referring to pure strains and the corresponding small letters to the
hybrids from the same species of egg. (/) refers to pure bred F.
heteroclitus; (/) to the hybrid strain from the eggs of F. hetero-
clitus and the sperm of F. mayjalis; (77) to the hybrid from the eggs
of F. majalis and the sperm of F. heteroclitus; and (/) to pure
bred F. majalis.

In the main body of the paper certain abbreviations will be
used without further explanation:

For the pure F. heteroclitus strains the terms: pure heteroclitus,
H pure, or simply (#7) will be used.

For the hybrid strains from F. heteroclitus eggs and F. majalis
sperm, the terms: hybrid heteroclitus, H hybrid, or simply (/),
will be used.

For the pure F. majalis strains the terms: pure majalis, MM
pure, or simply (JZ), will be used.

For the hybrid strains from the eggs of F. majalis and the sperm
of F. heteroclitus, the terms: hybrid majalis, M hybrid, or simply
(mm), will be used.

The Process of Heredity 51

“I

III DESCRIPTION OF EXPERIMENTS AND PRESENTATION OF DATA
Data Derived from the Study of Living Material
1. Type Series (Series 1) (Plates II, III and IV)

This series was started at 2 p.m., on July 2, 1907. Fresh lots
of fish of both species were brought in and the conditions for the
experiment were practically ideal.

The stages of development earlier than those presented in the
table, as well as those later than the seven day period (the last
stage pictured in the table), will be described verbally or with the
aid of occasional isolated illustrations.

Conditions earlier than Stage 1 (18 hours). a Comparative
fertility of eggs to sperm of their own and that of the foreign species.

Out of 121 eggs of H pure, 108 cleaved (89 per cent fertile).

Out of 136 eggs of H hybrid, 84 cleaved (61 per cent
fertile).

Out of 92 eggs of M pure, 82 cleaved (88 per cent fertile).

Out of 103 eggs of M hybrid, 57 cleaved (45 per cent
fertile).

In both cases the eggs were more fertile to sperm of their own °
than to those of foreign species.

b Comparative rates of cleavage of pure and hybrid strains.
After two hours nearly all of the H pures and the H_ hybrids had
cleaved and were in the two-cell stage. About twenty minutes
later over half of the eggs in these two strains were in the four-
cell stage. Rapid counts of blastomeres of stages up to the sixteen-
cell stage convinced me that there was no appreciable difference
in the rates of early cleavage in pure and hybrid strains. Three
hours after fertilization (one hour later than in the heteroclitus
strains) both majalis strains had cleaved and were in the two-
cell stage. They also showed equal rapidity in passing to the
four, eight, sixteen-cell stages.

Other stages up to Stage 1 (18 hours) were passed over in the
night. No observations were made during this period.

Stage 1 (18 hours) (see Plate II): Ocular micrometer measure-

518 Jak. lal Newman

ments of the blastodiscs of twenty eggs of each of the four strains
gave the following figures:

EP pures(Hir): 16, 18, 16, 17, 17; 15,19; 18, 10,075.07, eL0;
15,18, 18, 18, 17, 19, 17, 17. Average m7-) min:
Fighybrid (in): 13, 13; 15) 14, 145 155 11, 175 cl2 uO, Ay
13, 18 12, 12, 13, 14, 15, 10, 14.. Average tami.

M hybrid (nz): 14, 14, 16, 17, 12, 12; 13; 18, TOs alysis

II, 14, 14, 14, 15, 15, 16, 18, 18. Average 15 — mm.
M pure (M7): 15,10, 15, 15, 10,17, 14,0055, 155,15 5)155 05,
Gy 105,15, 10, 16, 14; 13,14... Average m5 -pamme

These measurements were made in less than half an hour
in the order listed. The H hybrids had about fifteen minutes
advantage in time over the H pures and yet show a lower average
blaseodise diameter. A higher degree of variability is also notice-
able at this early period. The measurements of the two mayalis
strains show no marked differences between them. ‘The slightly
greater average diameter of the W pures might be due to the
time advantage of about fifteen minutes that elapsed between the
measurements of the two strains. After determining the average
diameters of blastodiscs in all four strains camera drawings
of an average embryo of each strain were made and are reproduced
in the table. The measurements made with the ocular microm-
eter, when compared with a micrometer scale on the stage,
showed a magnification of 17 diameters, so that, in order to reduce
the measurements to actual millimeters the figures must be divided
by 17.

The difference in the average diameters of blastodises between
HT pure and H hybrid was so marked that it must have been
evident for some time.

Stage 2 (24 hours) (Plate II): The majority of the H pure
embryos showed the germ ring nearly halfway around the yolk
and a fairly well defined embryonic shield. The condition seen in
practically all of the specimens of H hybrids was distinctly less
advanced than in the 7 pures, the blastodisc still forming a
shallow cap over the yolk mass. No measurements were made of

The Process of Heredity 519

this condition since it was sufficiently obvious without measure-
ment (Plate II, H2 and h2).

In M hybrids ocular micrometer measurements of the diameters
of twenty blastodiscs were as follows: 21, 20, 23, 19, 21, 21, 19;
2331105 20920,22,,21,, 10, 17,,20;.21,,20, 22,18. Average\20:25 mm.
(Plate II, m2).

Twenty M pures gave 18, 16, 16, 17, 18, 16, 17, 17, 17, 18, 18,
16, 17, 18, 17, 16, 18, 16, 18, 17. Average 17 mm. (Plate II, M2).

The M pure and M hybrid embryos seemed to be about eight
hours behind the H pure and H hybrid embryos respectively.
The M hybrids showed a distinct and measurable advantage over
the VM pures of the same age. According to measurements, then,
the two pure strains show the extreme differences in time rate of
development, while both of the reciprocal crosses show inter-
mediate condition.

Stage 3 (48 hours): Nearly all of the H pures showed the con-
dition figured in Plate I] (H3). The optic vesicles were large
and showed the cavity plainly. Three or four mesoblastic somites
were visible on practically all of the specimens examined. Out
of fifty-five specimens examined two were noticeably retarded,
showing much smaller optic vesicles without a visible cavity,
and no mesoblastic somites.

The H hybrids showed a wider range of variability. Of the
forty-seven examined seven or eight presented a retarded condition
similar to that of the two retarded specimens of H pure. Six
showed a condition still less advanced, resembling (773), but with
the blastopore closed. No somites were visible on any of the
specimens. On the whole it was an easy matter to determine
that the H pures were considerably in advance of the H hybrids.

With regard to the VW pures and the M hybrids it was a matter
of considerable difficulty to determine which of the two strains was
in the lead (Plate II, m3 and M3). A fairly large per cent of the
M hybrids, however, showed a condition more advanced than any
M pures. One might be justified, then, in claiming that the hybrid
strain was in advance of the pure. Other series, as will be seen,
showed a much more marked difference between these two strains
at the same stage of development.

§20 H. H. Newman

Stage 4 (54 hours): The H pures showed a very uniform con-
dition, like (H/4). The lens was well developed and the optic
cup was invaginating. ‘The optic vesicles were clearly defined.
The primary interbrain was slightly lobed. There were on all
healthy specimens examined about twelve somites. A few very
much retarded specimens occur (Plate III, H4).

The H hybrids were markedly behind the H pures. The
most advanced condition resembled closely (473), but the majority
were like (hg). About 25 per cent resemble (h3). The most
advanced specimens had only four or five somites (Plate III, /4).

The M hybrids showed very little difference from the M pures so
far as the overgrowth of the germ ring and closure of the blastopore
were concerned, but the optic vesicles of the former were con-
siderably better developed than those of the latter. The condition
of the average M hybrid was scarcely more advanced than (h3),
(Plate III, m4).

M pures show a condition less advanced than M hybrids, between
(h3) and (m3). M pures were from eight to ten hours behind H
pures at this stage (Plate III, 174).

Stage 5 (72 hours): The H pures had taken a very marked
lead upon the 1 hybrids, for which the earlier establishment of a
heart-beat, with the accompanying vitelline and body circulation,
was probably responsible. The various brain lobes were large
and well defined. ‘The optic cup was fully invaginated and the
lens spherical. The embryonic eye showed a considerable cavity
between the lens and the inside of the optic cup. Accompanying
the establishment of a circulation a large amount of pigment had
been laid down in the form of dark brown chromatophores, scat-
tered over the body and the yolk mass. On the latter they originate
in connection with the capillaries. “The trunk had become opaque
with pigment. ‘The embryos had, as a rule, quickened, wriggling
of the tail region being very noticeable. The rates of heart-
beat in ten specimens were as follows: go, 98, 96, 92, 98, 97; 95,
98, 99, 95. Average, 95.9 beats per minute (Plate III, H5).

The H hybrids (Plate III, 45) were not nearly so advanced as
the H pures, showing a condition only slightly more advanced
than that shown by the #7 pures at fifty-four hours (H4). Of

The Process of H eredity 521

forty specimens examined four showed the feeble beginnings of
a heart-beat, which was slow and irregular and at the rates of 26,
36, 44 and 42, an average of 37 beats per minute. ‘These four
specimens and some others were somewhat larger in size than the
rest and showed a slight differentiation of the primary brain
vesicles. The optic vesicles were well cupped and the lens
spherical in all, but there was marked variation in the degree to
which the heads of the various embryos had developed. The
auditory vesicles were in all well defined. There were many
somites, usually over twenty. In none of the specimens was
there any pigmentation either on the body or on the yolk. In
none had quickening occurred.

The M hybrids were at this time well in advance of the MW pures.
The average condition is figured in the table (775). The head
region of the majority of the M hybrids showed a condition even
more advanced than that of the other hybrid strain (15). There
was a marked outlobing of the inter-brain. The optic vesicles
were well cupped, the lens spherical and embedded in the cup.
Auditory vesicles were well defined. There were fifteen somites,
on the average (Plate II, 75).

The M pures were less advanced, showing a less well developed
head region, and an average of about nine somites. No pig-
mentation present on either of the majalis strains (Plate III, 1/5).

Stage 6 (80 hours): During this rather short daylight period of
six hours, from 2 to 8 p.m., on a warm day, a considerable change
in the interrelationships of the four strains was apparent. The
H pures had advanced slightly. The heart-beat had increased
to an average of 110 beats per minute in ten specimens. The
pigmentation was considerably heavier and the size of the embryos
had increased noticeably (Plate III, H6).

In the H hybrids, however, a remarkably rapid change had
occurred. The establishment of a heart-beat and a circulation,
early in the afternoon, had caused a very rapid advance and at
this time the H hybrids were nearly as well advanced as the H
pures. A few specimens were quite as advanced as any of the 1
pures. The rates of heart-beat in ten examined were: 100, 96,
go, 88, go, 98, 92, 99, 90, 80. Average g2+. No pigmentation

522 H. H. Newman

Was as yet apparent in any specimen, in marked contrast to the
condition seen in the H pures where the pigmentation was readily
visible to the naked eye. The head region of the average H
hybrid was noticeably less advanced than that of the average H
pure (Plate III, 46). In some few specimens the embryos were
still as small and undeveloped as were the average H hybrids at
72 hours (h5). These retarded specimens numbered about to
per cent of the whole.

The M hybrids were still in advance of the WM pures, but the
difference was not so marked. In both of the majalis strains
the number of somites had increased to such an extent that it
was difficult to count them. ‘The average size of the MW hybrids
was a little greater than that of the WV pures, but this difference
was not well marked. The VW hybrids had begun to lag in devel-
opment and the W pures had almost overtaken them (Plate III,
mo, and M6).

Stage 7 (96 hours): The H pures had advanced chiefly in the
acquisition of a more definite body outline. Pigmentation had
become heavier and had rendered the body rather opaque,
making it difficult to see the outlines of the brain and nervous
system. The optic cavities were more pronounced and _ the
eye had become slightly pigmented (Plate IV, H7).

The H hybrids (Plate IV, 47) had grown to be a little larger
on the average than the H! pures, but were comparatively pale,
only a very faint pigmentation being visible. This failure to
pigment at an earlier stage may be due to the fact that the paternal
species does not show any pigment until several days later than this
period. The heart-beats of ten numbered as follows: 126, 120,
TOs OO,e112, 124, 120, 110,128, 110. Average 114.2 beats per
minute. ‘This average is about seven beats to the minute faster
than the average of ten H pures of the same age. The rates of
heart-beat of all but one retarded specimen, with a rate of 68, are
considerably faster than those of the latter. The specimens with
beats of 126, 120, 124, 120, 128, were especially large and were
noticeably more heavily pigmented than the others, although none
of them were nearly so dark as the average H pure.

The M hybrids, in general body form, seemed to be about on an

The Process of Heredity 523

equality with the M pures. Heart rates of ten gave the following:
84, 82, 94, 83, go, 82, 86, 66, go, 78. Average 83.5. The average
state of advancement was about equal to that of H pures at 72
hours (H5), (Plate IV, ™7).

The M pures (Plate IV, M7) appeared very like the M hybrids.
The heart rate, however, was very slow and feeble as yet, averaging
42 in ten specimens. Evidently the heart rhythm in M hybrids
was established considerably earlier than in M pures. All embryos
of both majalis strains were at this time entirely devoid of pig-
ment. ‘To the naked eye they appear very pale as compared with
the heteroclitus embryos.

Stage 8 (114 hours): The past eighteen hours showed a marked
advance in the H pure embryos. They had rounded out and
now exhibited a typically fishlike form, with caudal fin well
defined, the whole body opaque, and the eyes pigmented. The
figure (F8) will show the general appearance better than a verbal
sesomnion Large chromophores were present on the head
directly over the Bee and upon the surface of the eye. The yolk
mass was shrunken to a noticeable extent (Plate IV, HS).

The H hybrids (Plate IV, iS) were on the average not so ad-
vanced as the H/ pures, exhibiting a condition more like that of the
H pures at 96 hours (#717), except that many of the H hybrids were
larger than the latter. “The pigmentation was as yet considerably
lighter than in the pure strain. The heart rate of ten H hybrids
averaged 128 as compared with 112 for the 7 pures of the same
age. More retarded and ill-developed specimens were noticeable
than at any previous stage of development, probably on account
of the failure of many to establish a normal circulation. A num-
ber of such ill-developed embryos were no farther advanced than
the average H pure at 72 hours (H5). On the other hand
there were a few rather precocious specimens that were nearly
as advanced as the average H pure of the corresponding stage
(H8).

The majority of the M hybrids (Plate [V, mS) were in size and
general structure about like the M pures. In certain details,
however, they were very different. The majority were lightly
pigmented both on the body and the yolk. The heart rates of ten

524 lel. 15h Newman

were as follows: 118, 112, 112, 104, 110, 110, 120, 110, 114, 98.
Average 110.8. A few poorly developed specimens were noticed,
resembling the condition of MW hybrids at 80 hours (m6). About
go per cent, however, were large and healthy looking.

The M pures were about the size of the best of the MW hybrids.
About 10 per cent, however, had died since the last examination.
There were several ill-developed and anemic specimens. The
heart rates of ten specimens were as follows: 82, 80, 78, 78, 80, 82,
80, 80, 82, 82. Average 80.4. Although there was a complete
vitelline circulation there was no pigment on any of the specimens.
The heart-beat in the M pures was nearly two days later in appear-
ing than in the H pures. The pigmentation was still more mark-
edly slower in making its appearance (Plate IV, WS).

Stage g (168 hours or 7 days): Several stages were examined
between the last stage described and the present one, but there
was no very noteworthy change in the relative conditions of the
four strains. At this period the H pure embryos had grown con-
siderably in size, the eyes had reached almost their definitive form
and were darkly pigmented. ‘The pigmented areas were well
marked and consisted of large chromatophores. The pectoral
fins had appeared and were in a continual state of vibration, prob-
ably performing a respiratory function. The heart-beats were
difficult to count on account of the opacity of the body, but trom
special studies on other series it appears that the rhythm estab-
lished at Stage 8 is maintained at least up to hatching. The
capillaries and vitelline vessels were covered with brownish black
pigment. Larger chromatophores of a lighter brown color were
found between the capillaries.

The H hybrids (Plate IV, i¢) were on the average equal in size to
the H pures (Plate IV, HQ), but as a rule were much less heavily
pigmented, many being decidedly pale. About ro per cent of the
hybrid embryos were, however, at least as heavily pigmented
as any of the H pures, and some of these dark hybrids were notice-
ably larger than the largest of the H pures. The poorly developed
specimens showed all degrees of advancement. Some of them
lacked a circulation, although the heart was present and was beat-
ing at a fairly high rate. The hearts of some were shrunken

The Process of Heredity 525

and bloodless, mere strands that continued to contract rhythm-
ically within an enormous empty pericardium. A number of
these abnormal conditions will be described and discussed in
a later section of this paper. The hybrids, although a hetero-
geneous lot, still showed about 60 per cent of normal, healthy
specimens.

The M hybrids were too obscure within the envelope to admit
of accurate camera drawings. A typical specimen, however,
was dissected out of the envelope and, after being quieted with
chloretone, was drawn (Plate IV, mg). The general body form
is similar, except that it is larger, to that of the average H pure
of 114 hours (HS). The caudal fin was well developed and the
eye had assumed a form almost definitive. Heavy blackish pig-
ment, in the form of large chromatophores, had darkened the sur-
face of the yolk sac, while slender grayish, much branched chro-
matophores were distributed over the surface of the body.

The M pures (Plate [V, Mo) were on the average about the same
in size as the M hybrids. ‘There was a small amount of blackish
pigment on the yolk, but none at all on the body.

The verbal description of the stages figured in the table ends
here. The further description of the series will be carried on with-
out numbering the stages. ‘The sections, instead of being num-
bered by stages will be headed with the number of days that have
elapsed since fertilization.

At 10 days: The general size of the H pure and H hybrid
embryos was about the same, the chief difference between the
two strains being that the healthy H hybrids were a little
more heavily pigmented than the average H pures. A few
H{ hybrids were especially dark. ‘The M hybrids showed a heavy
pigmentation on the top of the head and down the middle of the
back. he vitelline vessels were also very heavily pigmented,
about like that of the average H pure at 96 hours (H7). The
general size of the WM hybrids was less than that of the W pures.
There had appeared a change within the last two days in the M
hybrids. “They seemed to have lagged behind the pures after
having been at first ahead of the latter and, for some time past,
practically on an equality with them. The M pures although

526 H. H. Newman

now noticeably larger than the M hybrids, were not nearly so
heavily pigmented. A certain degree of pigmentation had appeared
since the last stage described, but the color was still hardly visible
to the naked eye. The chromatophores of M pure were small-
bodied and much branched, quite different in appearance from
those seen on the H pures and the two types of hybrids. The
chromatophores of these three strains are practically alike. They
are much thicker bodied and less branched than those of M pure.
In other words, the type of chromatophore of F. heteroclitus
seems to be dominant.

At 11 days: The H hybrid embryos, with the exception of
about 25 per cent which were ill-developed and obviously unhealthy,
showed heavier pigmentation than the average H pure. The
chief reason for this seemed to be that the pigment of the paternal
species (F. miajalis) is blacker, even if more diffuse, and, when
aggregated in denser masses as It is in the hybrid, it gives a darker
coloration. The H hybrid embryos were on the average larger
than the HT pures and the egg membranes seemed to be under
some tension. The M hybrids seemed to have stopped growing,
while the 7 pures had advanced rapidly. ‘There was at that time
a marked disparity in size between the M pure and M hybrid
strains, the former being about 50 per cent larger than the latter.
A few unhealthy M pure embryos were pasta at this time.

At 12 days: 24 H pures hatched out during the forenoon of this
day, before any change in the water had been made, and hence
without any artificial stimulus. A camera drawing of one of the
best of these was made on hatching, the specimen being quieted
with chloretone (Plate V, Fig. H). The figure shows a dorsal
view. During the first four hours of the afternoon twenty-five
more H pures hatched, along with two specimens of H hybrid.
In almost every particular these two hybrids resembled the H
pures that had just hatched. “They were as darkly pigmented
as the darkest specimens of the H pures, were a little larger than
the average H pures, but not noticeably so. The color pattern
on the head and back was identical with that on the H pures.
These two hybrids were also as early to hatch as the average H
pures, and hatched without any artificial stimulus. The M hybrids

The Process of Heredity 527

had evidently ceased to develop. The largest specimens had
reached a size almost equal to that of the average H pure on hatch-
ing. The color pattern of the head and back was almost identical
with that of the two heteroclitus strains on hatching. The yolk
was very heavily pigmented and was still a large mass. The M
pures were on the average nearly twice as large as the WM hybrids.
The body was pigmented with large oven chromatophores.
The yolk was lightly pigmented with Biel

At 13 days: About fifty more H pures hatched. No more
hybrids hatched in spite of the water being changed and the conse-
quent stimulus afforded by the mechanical disturbance and the
sudden change of temperature. MM hybrids and M pures in
relatively the same condition as on the previous day.

At 14 days: Practically all of the H pures hatched. Only
few anzmic specimens left unhatched. No more H_ hybrids
hatched, but they had become more heavily pigmented, especially
on the yolk. A large specimen, with heavily pigmented yolk,
was dissected out of the egg membrane and it lay almost inert in
the sea-water, showing that it was not ready for hatching. The
length of this specimen was 7.5 mm., while that of the first H
hybrid hatched was 7.2 mm., and that of the first hatched H pure
was 7.3 mm. This embryo then although not nearly ready to
hatch, was somewhat longer than the hatched embryos of either F.
heteroclitus strain. ate in the evening of the same day six
more H hybrids hatched without artificial disturbance. M pures
greatly increased in size, but M hybrids no larger than at the last
observation.

At 15 days: Fifteen more H hybrids hatched. A typical speci-
men of this lot was quieted and drawn (Plate V, Fig. h.)

At 16 days: Twenty-two more H_ hybrids hatched. Only the
abnormal specimens, that could never hatch, were left within the
membranes. ‘These latter number about 25 per cent of the whole.
The H hybrids hatched out during the day were not nearly so
heavily pigmented as those that hatched out earlier. They were
also somewhat sluggish on hatching, some of them appearing to
be cramped by over-long confinement within the egg membrane.
A considerable number of those that hatched on this day did so

528 Ee lel; Newman

only on the application of some stimulus, such as squirting
them violently out of a pipette or pricking the egg membrane
with a sharp needle. Such specimens lack the initiative to hatch
without some assistance and would have died within the egg mem-
brane had not assistance of some sort been given them. When
released these individuals swim about rather sluggishly, and,
on being transferred to aquaria are very apt to float on the surface
and allow themselves to be drawn by the currents to the gauze
around the standpipe, and thus perish. Active young fish, such
as the previously hatched H hybrids, always go to the bottom and,
if drawn by a current, swim actively away from the region of
danger.

7 2

Figs. 1 and 2 show comparative sizes of M pure and M hybrid embryos at nineteen days (Series I).

Fig. I represents an average specimen of M hybrid.

Fig. 2 represents an average specimen of M pure.

The figures are camera drawings of embryos as they lay within the egg membrane, quieted with
chloretone. Figures show a magnification of 12 diameters.

At 19 days: No more H hybrids hatched, but many still living.
The M pures had grown considerably and looked as though they
were almost ready to hatch. A fairly heavy deposit of grayish
pigment covered the body but was sparingly scattered on the yolk
mass. The M hybrids showed no change except that they had
grown darker. Outline camera Aa of typical embryos of
the two mayjalis strains at this stage will emphasize the very marked
difference between them (Figs. 1 and 2 ).

At 22 days: About twenty M pures hatched. A typical speci-
men, drawn on hatching, is shown in Plate V, Fig. M. This
specimen was II mm. in length as compared with 7.2 mm. for the H

The Process of Heredity 529

pure drawn on hatching. The head diameter of this M pure was
1.8 mm., as compared with 1.3 mm. for the H pure. Two more
H1 hybrids had hatched since the last examination, but were dead
when noticed. Both specimens were pale, twisted and emaciated.

At 23 days: About thirty more M pures hatched. Nearly all
of the rest were dead, many of them having reached almost the
maximum size before dying.

At 27 days: All embryos of the series except about thirty H
hybrids and about sixty M hybrids, were dead. The H hybrids
showed a wider range of developmental stages and abnormal con-
ditions than the M hybrids. The latter were a fairly healthy
looking lot, seemingly quite normal except for the presence of the
large yolk mass attached to them. Some of them, when liberated
by dissecting off the egg membrane, swam about quite actively
and continued to live for several days in sea-water, although
handicapped by the presence of their abnormally large yolk sacs
full of a kind of material that they seem utterly unable to assimilate.
I tried to free them from their encumbrance by pricking the sac and
liberating the excess yolk, but the specimens thus operated soon
died, probably from loss of blood, since some blood was seen to
escape when the yolk material issued from the sac. A number of
the M hybrid embryos, liberated artificially from the egg mem-
brane, lived for as long as four days, but during this time the yolk
mass did not diminish and the embryos probably starved. A
typical specimen of these artificially liberated embryos was quieted
and drawn (Plate V, Fig. m).

All of the H! hybrids were dissected out of their membranes and
presented a bizarre collection of freaks. A number of the types
shown were drawn and will be seen in Figs. 3 to 8 inclusive. Fig.
3 presents the type most nearly normal. Fig. 4 is an unusual
type, showing an enormous growth of pectoral fins, which seem to
have continued to grow although the rest of the body had ceased to
increase in size. This might be explained by the fact that the
only noticeable circulation in the whole specimen was in these fins.
Figs. 5, 6 and 7 show various degrees of a common type, in which
the trunk region was greatly reduced. Fig. 8 shows a rare type,
in which the head region was ill developed, although the trunk was

530 ele Jele Newman

almost normal. In none of the specimens examined was the circu-
lation normal. In some it was partial, while in the majority it
was totally lacking, except in the heart itself. Much of the ill
success in development was probably attributable to some lesion
in the circulation, for which a reason will be suggested in the dis-
cussion.

Figs. 3 to 8 (inclusive) represent types of monstrosities in the H hybrid strain, dissected out of the
egg membrane, after all specimens capable of hatching had done so. These are camera lucida draw-
ings, showing a magnification of 12 diameters.

Fig. 3 represents a common type, well advanced, but with some lesion in the circulatory system.

Fig. 4 represents an unusual type, in which the pectoral fins and the eye have continued to develop
independently of the cessation of growth in the rest of the body.

Figs. 5, 6 and 7 show types of the tailless monster, the most frequent abnormality.

Fig. 8 represents an uncommon type, a headless monster. The head region is represented by a very

small, shrunken, heavily pigmented mass.

Considerable independence was seen in the development of
different regions. Head may develop while trunk is retarded
and vice versa. The fins in one case had continued to develop
after the rest of the body had ceased to grow.

A considerable number of H hybrids of this series are at the
present time being reared in aquaria at the United States Bureau
of Fisheries at Woods Hole, Mass.

The Process of Heredity 531

The data of five other series, almost as complete as the one just
described in detail, are on hand and furnish many interesting
comparisons and some new facts. There was a_ surprising
uniformity of results in the three other series conducted at about
the same time as the type series. After the development of a
method the results of all experiments that were carefully performed
were as similar as could be expected. Some of the earlier series,
however, gave results that varied widely in some particulars from
the type described. The plan followed is to take up first the series
in which all precautions were used, presenting any new or inter-
esting data as briefly as possible. Then will follow a very brief
statement of the results of earlier series in which no special effort
was made to equalize conditions of parent or embryos. Following
that will come a statement of confirmatory or contradictory results
of incomplete or partial series.

2 Other Series

Series 2, Ffuly 5, 1907. This was more like the type series
than was any other complete series. | The experiment was, how-
ever, conducted in a somewhat different manner. Fresh lots of
fish were used and after three hours twenty eggs that had success-
fully cleaved were selected from each of the four strains. No
careful counts of blastomeres were made but it was noted that there
was no distinct difference in time rate of cleavage between the
pure and hybrid strains.

At 14 hours: Ocular micrometer measurements of blastodisc
diameters of all embryos, gave the following averages: H_ pure,
.88 mm.; Hf hybrid, .87 mm.; WM hybrid, .gt mm.; M pure .89 mm.
The differences were too small to be significant and it is’ probable
that the influence of foreign sperm had not made itself felt to a
measurable extent as yet.

At 19 hours: Average blastodisc diameters of all eggs: H pure,
1.27 mm.; Hf hybrid, 1.12 mm.; M hybrid, 1.08 mm.; M pure,
1.11 mm. Here the difference between the H hybrids and the
pures was quite marked, but that between the two mayjalis strains
was still very slight.

At 22 hours: Camera drawings of each of the embryos were

532 H. H. Newman

made and afterward measured in order to see what proportion of
the yolk had been surrounded by the germ band. The results:

H pures, 24.6 per cent of yolk covered;

H hybrids, 20.8 per cent of yolk covered;
M hybrids, 18.2 per cent of yolk covered;
M pures, 16.2 per cent of yolk covered.

At 30 hours: An interesting condition between Stages 2 and 3
of Series 1. Here the H pures had the blastopore almost closed
and the optic lobes well developed, resembling (h3). The H
hybrids showed the germ band about three-fourths around the
yolk and the embryo in the primitive streak condition, slightly
more advanced than (73). M hybrids with germ band about
one-half around the yolk and a germinal shield better developed
than in M pures. Germ band in M pures less than one-half
around the yolk.

The rest of the series offered only a few noteworthy differences
to the conditions seen in Series I.

A tabulated account of these differences follows:

a At 56 hours there was no marked difference in degree of
advancement between M/ pures and M hybrid, in both the con-
dition resembling closely (V4).

b At 70 hours, however, the M hybrids showed a marked
advantage over the M/ pures, the former resembling (75) and the
latter (M5).

c At the end of a week the M/ pures and M hybrids were deci-
dedly clearer within the egg membrane and admitted of camera
drawings. The drawings show the two strains to have been
practically identical in size, but the M hybrids were heavily pig-
mented both on body and yolk, while the W/ pures showed no pig-
ment on the body and only a few finely branched, grayish chro-
matophores on the yolk.

d The H pures hatched as follows: 2 on eleventh day, 14
on twelth day, 2 on thirteenth day. The H hybrids hatched as
follows: 1 on eleventh day, 10 on fourteenth day, 4 on fifteenth
day,and 1 onsixteenth day. The remaining four showed decidedly
abnormal conditions like those, shown in Figs. 3 to 8, and never

The Process of Heredity 533

hatched. The first 11 H hybrids to hatch looked darker and
larger than the hatched H pures. The M pures hatched as fol-
lows: 4 on twentieth day, 6 on twenty-first day, 4 on twenty-sec-
ond day; the rest died. None of the M hybrids hatched. Eigh-
teen out of twenty of them were living on the twenty-fifth day and
micrometer measurements of head diameters were made for the
sake of comparison with those of the other strain on hatching.
The following were the measurements 1.28, 1.24, 1.24, I-14, I-45,
Ee24e 110s 1630) Neal4y, Lehn Ol ul 204) Tedae ln 30, 112, 1-0256.07) In24
Average 1.06+ mm. Average head diameters of M pures on
hatching was 2 mm.; that of H pures, 1.24 mm. Except for the
presence of three dwarfed specimens the average of the M hybrids
would have been a little greater than that of the H pures and the
specimen with head diameter of 1.45 mm. was larger than any of
the H pures. Even this large specimen, however, was not as
large as the smallest of the / pures measured.

Series 3, fuly 3, rg00. In this series no micrometer measure-
ments were made, but as only ten embryos were followed for each
strain, it was possible to become personally acquainted with nearly
all of the specimens and judgment was based on this acquaintance.

At 25 hours: H pure, germ band one-half around yolk. H
hybrid germ band one-third around yolk. No difference between
majalis strains.

At 48 hours: H pure, between (f73) and (H4). Average
somites, 8.

H hybrid, slightly less advanced than (H3). No somites.

M hybrid slightly more advanced than (m4).

M pure, slightly more advanced than (M4).

At 62 hours: No very marked difference between H pures and
Hf hybrids. M pures and hybrids also almost on an equality. At
this time there seemed to be a halting on the part of the two strains
that had been ahead, giving an opportunity for those that had
been distinctly behind, temporarily to overtake them.

At 68 hours: Six out of ten H pures had slow and feeble heart-
beats. No heart-beats in any of the other strains.

At 76 hours: In size and general body form the H pures were

534 H. H. Newman

like (H's), H hybrids like (26), M hybrids between (m6) and (m7),
and M pures between (m5) and (m6). H pure highly piemened
and with the following heart rates: 58, 58, 74, 62, 48, 70, 42, 75,
70,56. Average 61.3. H hybrids no pigment and with the follow-
ing heart rates: 74, 42, 66, 48, 76, 48, 64, 60, 108, 72. Average
64.8. Mhybrids no pigment, following heart rates: 60, 48, 62, 60,
60, 52, 60, 56, 64 (one not yet beating). Average, exclusive of
the last, 59. pure no pigment and no heart-beat.
At 84 hours: Heart rates as follows:

H pure—78, 78, 84, 56, 78, 72, 72, 74, 80, 82. Average
Tot:
H hybrid—84, 74, 74, 80, 90, 78, 78, 94, 92, 94. Average
83.8.
M hybrid—88, 80, 96, 84, 86, 92, go, 88, 84, 86. Average
87.4.
M. pure—72, 62, 66, 66, 64, 60, 68, 72, 64, 60. Average
65.4.
At 108 hours: H hybrids seemed somewhat larger than H
pures but were much more lightly pigmented. Heart rates as
follows:

HT pure—100, 108, 112, 100, 104, T12, 110, 102, 108; 100.
Average 105.6.

HT hybrid—116, 116, 140, 116, 112, 140, 142, 132, 152, 136.
Average 130.

M hybrid—120, 128, 120, 124, 124, 124, 36, 140, 136, 128.
Average 118.

M pure—132, 128, 132, 140, 144, 126, 132, 124, 140, 134.
Average 133

Other stages up to hatching not markedly different from Series
1. H pures hatched out on the average three days earlier than
H1 hybrids. M hybrids did not hatch at all, but reached almost a
uniform size, about equal to that of H pure on hatching. All
M pures hatched in an average of about twenty-two days.

Series 4, fune 23, 1¢07. This series was started with unusual
care and was followed for five days under close observation. It

The Process of Heredity 535

then became necessary to be absent from the laboratory for a few
days. After returning the experiment was not followed in detail,
but what records were taken show a striking resemblance to con-
ditions seen in Series 1. The figures made with camera lucida
during the first five days might almost be substituted, stage for
stage, for the figures in the table used for Series I.

Series 5, fune 19, 1906. Fresh adults of both species, at the
height of spawning season, used. Over 100 specimens of each
of the four strains.

At 52 hours: H pure lens formed and many somites, resembling
(H4). H hybrid no lens and few somites, slightly more advanced
than (h4).- M hybrids blastopore nearly closed and optic lobes
beginning to appear. M pure blastopore still wide open and no
optic lobes.

At 72 hours: H pures somewhat more advanced than (H5).
Pigment on body and yolk and hearts beating strongly. H hybrid
about like (m6). A few have very slow and feeble heart-beat.
No pigment. M hybrid between (73) and (H4). Lenses and
optic cups just forming. Auditory vesicles faintly defined. No
heart-beat and no pigment. pures less advanced than M hybrid
about like (#73), scarcely any trace of lens formation, no auditory
vesicles, and no pigment.

At 96 hours: H pures heavily pigmented. Hearts vigorously
beating. Embryos wriggling within the egg membrane. H
hybrids weakly pigmented in a few specimens. Heart-beat on the
average more vigorous and rapid than in H pures. M _ hybrids
heart-beat vigorous; no pigment; embryo wriggling. M_ pures,
heart-beats beginning to be established in a few specimens; no
pigment and no wriggling.

At 5 days: H pures very deeply pigmented with dark brown
chromatophores; average heart rate of ten, 109.7... H hybrid not
nearly so heavily pigmented as H pure; heart rate of ten, 113. M
hybrid as heavily pigmented as H hybrid; average heart rate of ten,
109. M pure, no pigment on body; extremely faint pigment on
yolk; average heart rate of ten, gg.

Other stages up to hatching were substantially like Series 1.
except that the series as a whole developed more slowly; the H

536 ETE Jab. N PLUITLAN

pures taking an average of eighteen days to hatch, the H hybrids
an average of twenty days, M pures an average of twenty-six days.
No M hybrids hatched.

The survivors of the three strains that hatched were placed in
balanced aquaria, were fed on clam juice, and generally carefully
looked after. The H hybrids throve in this environment and,
on the average, outgrew the H pures. | About a dozen of these H
hybrids continued to live in the aquaria after all of the H and M
pures had died. The survivors were transferred to an aquarium
provided with a standpipe, so that they could have a continual
flow of fresh sea-water, with the abundant supply of minute
organisms that are found in it. The young fish grew rapidly on
this diet and lived for seven months. During this time they were
looked after by Dr. Sumner, to whom I take this opportunity of
expressing my thanks for his kindness. Only four of these young
fish survived until March, at which time they simultaneously
sickened and died. A comparison of these young fish with pure
bred specimens of about the same age and size reveals an inter-
esting resemblance to both parent species. There is so little
material on hand, however, that it is deemed wise to reserve a
treatment of the advanced conditions until a greater degree of
success in rearing the young has been attained. It seems in no
way impossible to rear the hybrids to maturity.

Series 6, “fune 19, 1¢00. ‘This series was started on the after-
noon of the same day as the last series. Different parents were
used and only ten specimens of each strain (selected after cleavage
had begun) were followed. In all important details this series
agreed so closely with Series 5, that it seems superfluous to record
the data. It was, in fact, the striking resemblance between these
two series reared under the same conditions, that suggested the
idea of controlling future attempts at equalizing the developmental
conditions of the embryos by equalizing the physiological condi-
tions of the adults.

The six series above described are all that were studied in detail
through the whole or nearly the whole process up to the period of
hatching. Many other series, however, were started for other
purposes; to furnish material for experimentation and preserva-

The Process of Heredity 537

tion. Other series were conducted solely for the purpose of fur-
nishing quantities of young fish for rearing. Stull others were
followed for a while and then abandoned for lack of time to record
data, since it seems impossible to follow carefully more than about
three series at one time. Still other experiments gave results out
of harmony with the general trend of data. In fairness such
series should at least be mentioned, although it seems quite cer-
tain that the contradictory results were the outcome of poor method.
To distinguish these series from the more complete ones detailed
above, the various series will be designated by letter instead of
by number.

3 Fragmentary Data

Series A, fune 20,1906. Conditions of experiment not recorded.
Like Series 1 except that at 18 hours the H hybrids were noted
and drawn as being slightly more advanced than H pures. At
48 hours, however, the relative degrees of advancement shown in
Series 1 had been attained. “There seems to have been a retard-
ing effect due to foreign spermatozoa, the same in kind but differing
markedly in degree from that seen in other series. No further
observations.

Series B, Ffune 28, 1906. This was one of several series that
served to show the importance of using freshly taken fish. In this
case the fish used had been in aquaria for at least ten days and
had not been regularly fed. Only one observation was made—
at 24 hours—when the following data were obtained: About 50
per cent of H pures developing, the blastodiscs very small. Only
ro per cent of H hybrids developing, but these with blastodiscs
more advanced than the H pures. About 50 per cent of / hybrids
developing. Only about 5 per cent of W pures developing nor-
mally. The great majority of the blastodiscs were irregular,
cleft or ruptured. None of this strain developed to an advanced
condition. A few M hybrids reached a condition about like (78).

Series C, fuly 8, 1906. An incomplete series, not especially
contradictory. At 24 hours there was a much more marked
difference between H pure and H hybrid strains than shown in
Series 1. The blastodise of the H hybrids was about in the con-

538 ele H. Newman

dition of (Hr) while the germ band of H/ pures was on the average
more than halfway around the yolk. At 43 hours the blastopore
in H pures was nearly closed, more advanced than (m4), while in
H hybrid the germ band was less than one-half way around the
yolk and the embryo was very ill defined, between (H2) and (m3),
nearer the former. At 89 hours H pures resembled (m7); H
hybrid (m5); M hybrid (m5); and M pures (m6).

Series D, ‘fuly 2,1G¢06. This series was reared to hatch and was
observed only incidentally while the water was changed from
time to time. Just before hatching, however, counts of heart
rates were made with the following results:

Hf pure—troo, 108, 108, 106, 96, 110, 112, 120, 106, IIo.
Average 107.6.

HZ hybrid—t1oo, 130, 148, 120, 116, 100, 130, 120, 122, 124.
Average 121.

M hybrid—74, 110, 82, 96, 110, 116, 56, 100, 96, go.
Average 93.

M pure—1i2, 120, 124, 110, 126, 120, 120, 110, 128, 126.
Average 119.6.

In this series the heart rate of the two hybrids were the extremes
while the two pure breeds occupied a mean position.

Series E, “fuly 11, 1¢06. “This incomplete series was strikingly
in accord with Series 1 as far as it went, but was not followed after
42 hours, at which time drawings of typical specimens of all four
strains were made, H pure resembling (h4), H hybrid resembling
(m4) with blastopore almost closed, M hybrid resembled (H2),
and M pure resembled (h2).

Series F, fune 24, 1007. This series was followed carefully
for four days when it had to be abandoned, on account of
absence from the laboratory. At 48 hours this series showed the
unusual character of having the V/ pures decidedly in advance of
the M hybrids. At the end of three days, however, the M hybrid
had forged distinctly ahead.

Series G, Fuly 8, 1907. No observations recorded except at
time of hatching. H pures had all hatched at the end of fifteen
days while none of the H hybrids had hatched. Two days later
the majority of the latter had hatched.

The Process of Heredity 539

Series H, fune 23, 1007. H pures hatched chiefly on the
twelfth and thirteenth days. H hybrids hatched as follows: 10
per cent on the thirteenth day, 25 per cent on the fifteenth day,
30 per cent on the sixteenth day. The remaining 35 per cent did
not hatch. M pures hatched some time after the eighteenth day.
About thirty were seen swimming in the dish on the twenty-first

day.
Data Derived from the Examination of Preserved Material

Various series were watched and when some doubtful or espec-
ially interesting stage was reached a sufficient number of embryos
were killed in picro-acetic, and examined afterwards at leisure.
Additional and confirmatory data on several points was obtained
in this way.

1 Rates of Cleavage

1 The eggs of a large F. majalis female were divided into two
lots of approximately equal size and fertilized simultaneously
with the milt of the two species. After three hours development
eggs were killed and examined with the following results: M pure
showed 158 four-cell stages, 2 three-cell stages, 3 two-cell stages,
and four uncleaved. M hybrid showed: 89 four-cell stages, 2
three-cell stages, 1 two-cell stage, and 114 uncleaved.

2 Another lot of majalis eggs treated in the same way. M
pure 51 four-cell stages, 4 three-cell stages, 3 two-cell stages, and
38 uncleaved. M hybrid, 32 four-cell stages, 2 three-cell stages,
1 two-cell stage and 54 uncleaved.

3 Eggs of one heteroclitus female used, fertilized with both
species of sperm. After three hours there were: H pures, 41
four-cell stages, 5 two-cell stages, 54 unsegmented; H hybrid, 13
four-cell stages, 3 two-cell stages, 80 unsegmented.

4. Same as 3, but a large lot of mixed eggs from over a dozen
females were used. These were divided, after stirring thoroughly
into two lots and fertilized with two species of sperm. At the
end of three hours they were killed in picro-acetic acid, and
examined. Results: H pure, 308 four-cell stages, 63 two-cell
stages, 563 uncleaved. H hybrid, 214 four-cell stages, 18 two-
cell stages, 742 uncleaved.

540 H. H. Newman

There were probably many unripe and stale eggs in this lot, as
no care was takento select the best males or females.

5 A large number of eggs from a good H female were divided
into two lots, fertilized with both species of sperm, and after they
had reached a stage where there seemed to be about equal num-
bers of 16 and 32-cell stages the two lots were killed and examined,
with the following results: H pure, 38 thirty-two-cell stages,
44 sixteen-cell stages, and 29 uncleaved. HA hybrid, 33 thirty-
two-cell stages, 31 sixteen-cell stages, and 42 uncleaved. Of eggs
cleaved H pure showed 46.3 per cent in 32-cell stage and H hybrid
44.6 per cent. The difference was so slight that it was not sig-
ficant. Four other experiments similarly conducted gave results
approximately like those just detailed. In some the percentages
lightly favored the pure strain and in others the hybrid strain.
The conclusion was reached that there was no measurable differ-
ence in time rate of cleavage up to the 32-cell stage, at least.

6 Several experiments were conducted like those just described
except that the eggs were allowed to develop until the blastodiscs
had begun to spread out over the yolk. They were then killed
and examined before any shrinkage had taken place.

a_ ‘This lot allowed to develop for sixteen hours, and twenty-five
eggs, selected at random, were transferred to another vessel, where
the blastodisc diameters were measured with ocular micrometer.
Results: Hf pure average 20.4 mm.; H hybrid 16.6 mm.

b Allowed to develop 14 hours: Average diameter of twenty
H pures was 16.8 mm., that of H hybrids 15.4 mm.

c After 20 hours: Average diameter of twenty H pures was
22.6 mm., that of H hybrids 20.1 mm.

d After 20 hours: H pures 21.3 mm., H hybrids 20.8 mm.

7 This series only F. heteroclitus strains: After 24 hours the
two strains were killed and examined. It was found that the H
pures were on the average in the condition of (74), while H hybrids
were in that of (73). The difference was very marked.

2 Later Stages

A few detailed camera drawings of whole mounts of later stages
were made and a plate of these figures will show better than a

The Process of Heredity 541

verbal description could, how real a difference exists between
pure and hybrid strains at these stages (Figs. 9, 10, 11 and 12).

Set 1: Killed after 56 hours, ten embryos of each strain pre-
served. Numbers of somites:

HT pure: 10, 94, 104, 10, 10, 104, 10, 104, 94, 10. Average
10+. (Fig. 9.)

H hybrid: 54, 74, 6, none, 6, 4, 64, 64, 54,6. Average 5.3.
(Fig. 10.)

M hybrid: 6, 3, 2, 2, 3, the rest none. (Fig. 11.)

M pure: none in any of the specimens. (Fig. 12.)

The figures show an average condition in each case.
Set 2: Ten of each strain killed after 86 hours. Numbers of
somites:

EL pure 21, 21. 21.21, 20s 20-21, .210. 20a 2On aw AVeLage

20.8.

Hivhybrid:s1s,. 16; 17, 10, 15,910,904, 15, 17,120 average
153"

M hybrid: 14, 15, 12, 16, 16, 16, 16, 16,17, 16. Average
Wi a4

M pure—13, 13, 13, 13, 13, 13, 12, 13, 13,12. Average 12.8.

Set 3: Killed after 48 hours, ten embryos of each strain preserved.
As there were no somites another index of development was chosen.
Four stages of development were arbitrarily chosen and drawn
(Figs. 13, 14, 15 and 16). Then the various specimens were
referred to the stages by number. When the condition fell between
two numbered stages the figure 4 was used to express the inter-
mediate condition. ‘The results were as follows:

FT pure: 4, 34, 34 4) 34) 44) 14, 34) 4, 4. Average 3.6.
Hf hybrid 1, 2,24, 4, 24; 2,2, 24, 4, 4. “Average 2.65.
Mihybridis.2, 24,24, 25.1, 2, 1%, 1, 25 2, Ll. Averave 95.
ME PIE Mule D,, L5)To!2, 1%, Used. Average, Iol'c:

Several other sets showed practically the same facts as those
just detailed. Later stages do not make favorable whole mounts,
on account of their opacity.

542 H. H. Newman

Figs. 9 to 12 (inclusive) are camera drawings, showing a magnification of 20 diameters of the four
strains at 56 hours.

Embryos were killed and fixed in picro-acetic acid, dissected off the yolk, stained in borax carmine,
cleared in bergamot oil, and mounted in balsam.

Fig. 9, average condition in pure bred F. herteroclitus.

Fig. 10, average condition in hybrid F. heteroclitus 2 and F. majaliso’.

Fig. 11, average condition in hybrid, F. heteroclitus @ and F. majalis 9.

Fig. 12, average condition in pure bred F. majalis.

Figs. 10 to 16 (inclusive) are camera drawings of embryos prepared as in the series above. The four
figures represent the average conditions in four strains of another series at forty-eight hours. The
arrangement of figures and magnification is the same as in the series above.

The Process of Heredity 54

Experimental Data

The facts that the two species show such marked physiological
differences and that these differences exhibit themselves when the
embryos or adults are subjected to sub-optimal conditions, led to
experiments that would afford a comparison of the resistances of
the pure and hybrid strains at the same stage of advancement.
This type of experiment requires rather large numbers of specimens
and a considerable amount of repetition, on account of the high
degree of individual variation, especially among the hybrid strains.

t Resistance to Lack of Oxygen

In 1905 considerable time and effort were expended in the en-
deavor to determine the comparative resistances of the pure and
hybrid strains to lack of oxygen. Engelmann gas chambers, boiled
sea-water, and a continuous stream of purified hydrogen were used.
At that time it was not known that the resistance to lack of oxy-
gen varied with the degree of development, hence a mass of very
contradictory data was obtained. The following summer the
experiment was repeated several times with embryos of the same
age and degree of advancement. ‘The results were quite different
from what one would naturally expect. The H hybrids proved
to be the most resistant, the H/-pures next, the M pures next, and
the M hybrids the least resistant. The two hybrid strains were
the extremes while the two pure strains were the means.

These experiments were so tedious, necessitating watching for
nearly twenty-four hours, continuously, or at frequent intervals,
that another method of testing the comparative resistance of pure
and hybrid strains was tried. This method consisted of putting
the embryos in gas chambers divided into four compartments of
equal size, filling the chamber with sea-water charged with carbon
dioxide, sealing up the chamber and timing the cessation of heart-
beat in all specimens. Of course only small numbers could be
used, because it was necessary to keep a very close watch over all
specimens in order to get even approximately accurate death rates.

Experiment 1, fune 26, 1¢07. The M hybrids showed the

544 H. H. Newman

most immediate effect of the carbon dioxide, the heart-beat of all
five specimens ceasing before the expiration of 5 minutes. ‘The
M pures lasted, on the average 7 minutes. The H pures averaged
18 minutes. The H hybrids averaged nearly 30 minutes, all
continuing to live after all of the other embryos were dead.

Experiment 2, Fune 26, 1907. Six embryos of each strain,
eight days old. The M hybrids all stopped before any of the
others. The M pures began to show the effect of the carbon
dioxide a little later than the M hybrids and survived considerably
longer. The H pures stopped a few minutes later, while four out
of six H hybrids continued nearly twice as long as the H pures.

Experiment 3, fuly 8, 1907. Yen embryos of each strain used.
All 4 days old.

After 5 minutes, 6 M hybrids and 5 M pures dead.

After 10 minutes, 7 M hybrids and 5 M pures dead.

After 19 minutes, 8 M pures dead, one feeble and one
active.

After 21 minutes, 7 M hybrids dead, 3 very feeble.

After 28 minutes, all / pures and M hybrids dead.

After 33 minutes, 4 H pures dead, no H hybrids dead.

After 38 minutes, 6 H pures and 2 H hybrids dead.

After 45 minutes, 7 1 pures dead, 2 feeble, 1 fairly strong;
4 H hybrids dead, the rest strong.

After 50 minutes, 9 H pures dead, 1 feeble; 4 H hybrids
dead, 1 feeble, 5 strong.

After 60 minutes, all H pures dead; 5 H hybrids dead, 1
feeble, 4 strong.

After 75 minutes, 8 H7/ hybrids dead, 2 strong.

After 85 minutes, all H hybrids dead.

The experiment shows that, in embryos of four days, there was
practically no difference between the two mayalis strains, but that
the H hybrids were considerably more resistant than the H pures.

Experiment 4, ‘fuly ¢, 1907. An attempt was made to select
embryos of the same degree of advancement instead of the same
age. ‘The embryos were selected from different series and were

The Process of Heredity 545
about as advanced as the average H pure at 7 days. “Ten embryos
of each strain were used. Results:

After 5 minutes, 5 hybrids dead.

After 14 minutes, 1 / pure dead, 5 M hybrids dead.

After 16 minutes. 5 M hybrids, 1 M pure, and 2 H hybrids,
dead.

After 22 minutes, § M hybrids dead, 2 feeble; 2 M pures
dead; and 2 H pures dead.

After 36 minutes, 9 M hybrids dead, 3 M pures dead, and
2 H hybrids dead.

After 38 minutes, all M hybrids dead, 4 M pures dead and 4
feeble; 5 H pures dead.

After 46 minutes, / pure 6 dead and 4 feeble; H pures 8
dead; H hybrids 2 dead.

After 53 minutes, M pures and H/ pures all dead; H hybrids
6 dead and 2 feeble.

After 60 minutes, 8 H hybrids dead, 2 feeble.

After 65 minutes, all Hf hybrids dead.

This experiment shows that in strains of the same degree of
advancement the M hybrids were the least resistant, the M pures
next, the 7 pures next, and the H/ hybrids the most resistant.

Experiment 5. Only the two majalis strains used; ten embryos
of each used; six days old. The M hybrids were all dead before
any of the M pures; three of the MW pures survived any of the M
hybrids. :

Experiment 6. Only the two heteroclitus strains used; ten of
each; six days old. Results:

After 5 minutes, 1 7 hybrid dead.

After 8 minutes, 1 H/ hybrid; and 7 1 pure dead.

After 16 minutes, 1 Hf hybrid dead; 8 H pures dead, 2
feeble.

After 26 minutes, 1 H hybrid dead; 9 H! pures dead and 1
feeble.

After 35 minutes, 2 Hf hybrids dead; 9 H pures dead and
1 still feeble.

546 Jel. ak Newman

After 55 minutes, 8 H hybrids dead, 2 still vigorous, 1 H
pure still feebly living.
After 65 minutes all dead.

This experiment shows an interesting fact that there is a well
marked individuality among these embryos. One of the H
hybrids (the more resistant strain) succumbed before any of the
H1 pure (the less resistant strain); while one of the H pures (the
less resistant strain) resisted feebly as long as any of the H hybrids.
On the whole, however, the H hybrids showed themselves to be
the more resistant of the two heteroclitus strains.

2 Experiments with KCN

Believing that KCN acts as a reducing agent upon animal
tissues and produces the same effects as those produced by lack
of oxygen, rather strong solutions of KCN in sea-water were used
upon embryos and adults.

Experiment rt. Placed three adult males of each species in
a covered vessel filled with one liter of one-tenth molecular KCN
solution in sea-water. Results: The three F. majalis males died
in an average of 16 minutes; the three F. heteroclitus males died
in an average of 31 minutes. ‘The same experiment was repeated
with three females of the two species. Results: F. majalis females
died in an average of 18 minutes, while the F. heteroclitus females
died in an average of 30 minutes.

Evidently F. heteroclitus is much more resistant to KCN poison-
ing than F majalis.

Experiment 2. Three stx day embryos of each of the four
strains put into 75 KCN solution in sea-water:

The H pures lived for an average of 93.5 minutes.
The H hybrids lived for an average of 82.5 minutes.
The M hybrids lived for an average of 70 minutes.
The M pures lived for an average of 20 minutes.

Experiment 3. Same conditions as last experiment except that
ten embryos of each of the four strains were used. After 2 hours

T he Process of Heredity 547

in the solution they were all transferred to fresh sea-water and
allowed to recover in as far as possible. It was found that:

H1 pures 5 alive and 5 dead, 50 per cent living.

H hybrids 6 alive and 4 dead, 60 per cent living.
M hybrids 8 dead and 2 living, 20 per cent living.
M pures none alive and to dead, o per cent living.

The experiment shows no marked difference between the two
heteroclitus strains, but a more marked difference between the
two mayjalis strains.

Experiment4. Fifteenof eachof the four strains used. Embryos
seven days old. Same solution as in Experiment 3.

After 2} hours’ exposure to the poison the embryos were trans-
ferred to fresh sea-water and carefully washed. Sufficient time
was allowed for all not entirely dead to recover and then the dead
were counted:

H pures 12 alive and 3 dead, 80 per cent alive.
FH hybrids 6 alive and 9 dead, 40 per cent alive.
M hybrids 1 alive and 14 dead, 6 per cent alive.
M pures 4 alive and 11 dead, 26 per cent alive.

This experiment shows the effect on the heteroclitus strains
to be about the same as before, but the conditions for the majalis
strains are reversed, the pure strain being the more resistant.
The hybrids had begun to show the enfeebling effects of a slower
circulation, which finally results in a failure of all specimens to
hatch.

Experiment 5. “This experiment dealt only with the two hetero-
clitus strains. Embryos nineteen days old. Same solutions as
before. The H hybrids showed the first effects of the poison,
four dying before any of the H pures. Three H pures survived
all of the H hybrids.

The experiments with KCN show at least one thing, viz: that
the introduction of F. majalis sperm into the egg of F. heteroclitus
produces a hybrid with a lessened resistance to this particular
poison.

548 H. H. Newman
IV SUMMARY OF DATA

1 The volume of F. majalis eggs is on the average more than
twice that of F. heteroclitus. The rate of development is rather
closely proportional to the mass of the eggs, the smaller egg devel-
oping nearly twice as fast as the larger egg.

2 The yolk of F. majalis eggs is much more yellow and opaque
than that of F. heteroclitus. This optical difference undoubtedly
indicates a much more deep seated difference in chemical composi-
tion.

3. A far larger percentage of eggs are fertile to sperm of their
own species than to that of foreign species.

4 The rate of early cleavage is not measurably altered by the
introduction into the egg of sperm belonging to a more slowly or
more rapidly developing species.

5 The earliest measurable effect of foreign sperm in hastening
or retarding development is seen in the egg of F. heteroclitus. At
periods ranging from fourteen to twenty hours the blastodise of
HT pure shows a measurably greater diameter than that of H
hybrid. In the eggs of F. mayjalis this difference is not seen for an
average of about six hours later, but it is just as marked when it
appears.

6 In general the development of F. heteroclitus eggs is retarded
by the introduction of F. majalis sperm, while that of F. majalis
eggs is accelerated by the introduction of F. heteroclitus sperm.
This acceleration or retardation is not permanent for either of the
hybrid strains. The more fortunate of the H hybrids, although
retarded forthe first eight or ten days, at and subsequent to hatching
are somewhat larger, have a more rapid and more efficient circula-
tion, are more active in their movements, show a greater resistance
to lack of oxygen and the presence of carbon dioxide, and live
much longer in captivity, than do any of the H pures. The M
hybrids, on the other hand, develop more rapidly than do the MZ
pures for a period of from seven to ten days, but after that they
gradually cease to grow, attain a size only about half that of the
M pures at hatching (but about equal to that of the H pures at
the same period) and neyer succeed in hatching because of their

The Process of Heredity 549

apparent inability to consume the large mass of superfluous
yolk.

7 The heart-beatof the H pures appears about ten hours earlier
than that of the Hf hybrid and this gives the former a decided
advantage over the latter in rate of subsequent development.
When, however, the H hybrids attain a heart-beat and a circulation
it is a more rapid and efhcient one than that of the H pures (the
more rapid heart-beat being an endowment from the paternal
species), and they rapidly Berea the 1 pures and, for a time,
seem to be almost on an even footing with the latter, a small per-
centage of them certainly surpassing any of the H pures. The
heart-beat and circulation of M hybrids appears nearly a day
earlier than that of the / pures, and for a time the former show
a decided advantage over the latter. But when the M pures
attain a heart-beat and circulation it is a more rapid and efficient
one and the M pures overtake and pass the M hybrids. The
latter remain behind permanently and, with their slower heart-
beat and less efficient circulation they never succeed in consuming
more than half of the yolk with which the egg is endowed.

8 The phenomenon of pigmentation runs parallel with that of
circulation and is probably dependent to some extent thereon.
The H pure embryos become heavily pigmented in about three
days, while the pures become pigmented very late in develop-
ment, and then comparatively lightly. Naturally the H pures
show the first signs of pigmentation, dark brown chromatophores
appearing on yolk and body soon after the establishment of a cir-
culation. About a day later the H hybrids begin to show signs
of pigmentation, but at first far less abundantly dem the 7 pures.
In later stages, however, the most successful of the H hybrids
surpass in depth of pigmentation the most heavily pigmented of
the H pures. This may be explained by the fact that these hybrids
combine the F. heteroclitus character of densely packed chroma-
tophores with the F. majalis character of a darker colored pig-
ment. The. M hybrids show a much earlier and much heavier
pigmentation than do the / pures. ‘The difference at about five
days is very evident to the naked eye. ‘The color pattern on the
head and trunk, characteristic of F. heteroclitus embryos, is

550 Jaks tals Newman

found on all embryos of the M hybrid at the period of their maxi-
mum development. The same pattern is found on all successful
H hybrids embryos in advanced stages.

9 “The chromatophores of F. nelle are small-bodied and
finely branched, while those of F. heteroclitus are proportionally
larger bodied and much less finely branched. ‘The F. heterocli-
tus type of chromatophore is found in three out of the four strains,
viz: H pure, H hybrid, and M hybrid.

10 ©The size of F. majalis on hatching is about twice that of
F. heteroclitus. That of H hybrids is, on hatching, no larger, on
the average, than that of H pures. The maximum size reached by
the M hybrids is about equal to that of the H pures on hatching.

11 The examination of preserved material furnished more
easily measurable data for demonstrating the differences at vari-
ous stages of development between pure and hybrid strains. The
numbers of mesoblastic somites at various stages offers an accurate
and convenient measure of stages of development. In every case
examined the data derived from preserved material served to con-
firm observations made upon living material.

12 The hybrid strains, especially the H hybrids, showed a
remarkable degree of variability, far wider in range than was seen
in the pure strains.

13. In the experiments involving the lack of oxygen and the
presence of carbon dioxide, the two hybrid strains, instead of
occupying positions between the two pure strains, fell to the
extremes. The H hybrids were the most resistant, the H pures
next, the M pures next, and the MW hybrids the least resistant.

14 KCN solutions in sea-water were found to act in quite a
different way from carbon dioxide or lack of oxygen. Here the
hybrid strains seemed to occupy mean positions, although with
no great degree of regularity. The data derived from the use of
KCN was not very satisfactory.

The Process of Heredity 551

V_ DISCUSSION

The Relative Influence of Maternal and Paternal Elements in
Determining the Characters Seen in Early Development

Driesch,‘ as the result of his hybrid experiments with various
species of echinoids, came to the conclusion that the early develop-
ment of hybrids is practically determined by the character of
the egg protoplasm; that the rate of cleavage, the number of pri-
mary mesenchyme cells, the form of the larval body, and the color
content of the hybrid tissues, are exclusively maternal characters,
and that no male influence is felt until a comparatively late period,
when the larval skeleton is forming. These rather sweeping con-
clusions were subsequently modified to a slight extent in response
to a criticism by Boveri.* Driesch® admitted that at least one of the
characters claimed by him to be pure maternal, showed the male
influence. This was the character of intensity of coloration of the
hybrid larve. Further than that he was unwilling to go.

Boveri showed that the number of mesenchyme cells is notice-
ably influenced by the male parent. In fact, it appears from
Boveri’s work that the form of larval body and almost every char-
acter of the hybrids show the paternal influence. It was admitted,
however, that the chief controlling factor in early development is
the egg protoplasm.

Driesch and Boveri have never reached an entire agreement on
this matter. They stand for two opposite doctrines of heredity.
Boveri is a stanch champion of the idea that the nucleus is the
sole bearer of hereditary material and hence has a tendency to
overemphasize the paternal influence, since he believes the sperm
cell to consist almost entirely of nucleus. Driesch, on the other
hand, believes firmly that the cytoplasm 1s of equal value in inherit-
ance, and somewhat oversteps the mark in claiming that the egg
protoplasm is the sole factor in determining the character of the
phenomena of early development.

The data advanced in the present paper lead to a middle course.
For merely mechanical reasons the egg protoplasm must necessarily

4Driesch, H. Arch. f. Entw.-Mech., 7, 1898.

5 Boveri, Th. Arch. f. Entw.-Mech., 16, 1903.
® Driesch, H. Arch. f. Entw.-Mech., 16, 1903.

552 A. H. Newman

determine some of the characters of the hybrids, such as the
maximum size on hatching, the rate of early cleavage, and the degree
of transparency of the embryo and of the yolk sac. It is scarcely
to be expected that a mass of protoplasm and yolk, such as the
fish egg, should show an immediate measurable alteration on the
entrance of a body comparatively so minute as the fish sperm cell.
It naturally takes time for the sperm cell to reorganize the com-
paratively enormous mass of egg protoplasm to such an extent
that the rate of the developmental process is measurably altered.
As a matter of fact it should cause some surprise that the sperm
cell is able to do this work in so short a time as it does. The
data cited above show that there was a distinctly measurable
retardation of the developmental process in the case of H
hybrids after a period as brief as fourteen hours, a time very short
as compared with the total period of embryonic development. It
is practically certain that this influence of the sperm cell was
operative at a much earlier period, even though it was not measur-
able by the crude methods applied.

The only pure maternal characters that have been noted are
some of the characters that exist before the influence of the
sperm could reasonably be expected to make itself felt, and a few
mechanically determined characters such as have been mentioned.
In all other characters the sperm influence was evident in some
form or other.

These results and conclusions are out of accord with the results
of Loeb? and Godlewski,’ who found that the larva, produced by
fertilizing sea-urchin eggs with the sperm of holothurians and
crinoids, show d only sea-urchin (maternal) characters.

It seems necessary to take exception to a statement made by
Conklin® in a paper that appeared since this work went to press,
“that the early development of animals is of purely maternal
type, and that it is only in stages later than the gastrula, and con-
sequently after the broad outlines of development and the general

7 Loeb, J. University of California Publications, vol. i, 1904.

* Godlewski, E. Arch. f. Entw.-Mech., 20, 1906.
® Conklin, E.G. Science, vol. xxvii, no. 681, January, 1908.

The Process of Heredity 553

type of differentiation have been established, that the influence of
the spermatozo6n begins to make itself felt.’’ In the present work
I believe that the evidence shows that the influence of the sperma-
tozoon is probably immediate and becomes distinctly measurable
long before gastrulation.

Exclusive versus Blended Inheritance

Are there any examples of exclusive inheritance in the present
work? A pure maternal character, such as the rate of early
cleavage, might be termed a dominant character, but if we admit
it to be such, we have not far to go in search of a physical explana-
tion of this dominance.

Nearly all of the characters observed may be classed as exam-
ples of blended inheritance of one sort or another. The only
characters that cannot be thus classified, but seem to be akin to
exclusive inheritance are:

1 The size of the hybrids of both strains on hatching. The
size of the M hybrids at the period of maximum development,
which is the equivalent of the stage of hatching in the other strains,
is on the average that of the smaller species, F. heteroclitus,
although the hybrids have developed within eggs of F. majalis,
the larger species. "The H hybrids are on the average only equal
in size to the F. heteroclitus parent at the same period. The
character of size on hatching might be called a dominant F. hetero-
clitus character, for all strains containing F. heteroclitus are of
approximately equal size on hatching (see Plate V).

2 The color pattern on head and body of both reciprocal
crosses is, at a period shortly before and after hatching (in MW
hybrids at the period of maximum development) practically
identical with that of the H pure. F. heteroclitus might then
be called dominant with regard to the color pattern at this devel-
opmental period (see Plate V).

Another kind of inheritance occurs, which is neither exclusive nor
blended, but seems to be a sort of an exaggeration of exclusive in-
heritance, if we may be pardoned the expression. Cases occur in
which the hybrids carry certain characters to extremes, showing
them to a more marked degree than either parent species. We

554 H. H. Newman

might call such a condition hyper-dominance. Some examples of
this phenomenon follow:

1 When all four strains of a series were subjected to the experi-
mental conditions of lack of oxygen or the presence of carbon
dioxide, it was found that the H hybrids were the most resist-
ant, the H pures next, the M pures next, and the M hybrids
the least resistant. The H hybrids then carry resistance to
an extreme while the M hybrids carry lack of resistance to an
extreme.

2 A somewhat similar condition was seen in the degree of
depth of pigmentation of many of the H hybrids just before and
just after hatching. A considerable percentage of the latter become
more darkly pigmented than any of the H pures (the darker parent
species).

3 After hatching it was found that the more successful type
of H hybrids grew faster and lived far longer, under the conditions
applied, than any of the 7 pures (the parent species that lives
the longer under these conditions).

That there were all degrees of this dominance or hyperdomin-
ance cannot be denied, but the same can be said of practically all
cases of dominance described in the literature. There really
appears to be no valid reason why these cases just cited should not
be dealt with as examples of the phenomenon of dominance or
exclusive inheritance. [| think, however, that I can demonstrate
that these cases of dominance are the secondary physiological
results of the interaction of two sorts of blended inheritance, viz:
in the time of establishment of the heart rhythm and in the rate
of heart-beat, both of which affect the general circulation.

The heart rhythm of H pure, in accordance with the more rapid
development of this species, is established about 24 hours earlier
than in M pure. Both reciprocal crosses show an intermediate
condition, a blending more or less complete, with regard to this
character of time of establishment of heart rhythm.

In the case of rate of heart-beat the following are the facts: the
rate of heart-beat in F. heteroclitus, after it has been wellestablished,
is much slower than that of F. majalis, that of the former being on
the average about 135 beats per minute and that of the latter about

The Process of Heredity 555

105. ‘The individuals of both hybrid strains show all degrees of
blending of these two rates, each strain showing an average not
far removed from the ideal mean, 125 beats per minute.

The effects of these two cases of blending are far-reaching, and
are quite different in the two hybrid strains.

The H hybrid strain shows the F. majalis influence in acquir-
ing a heart rhythm about ten or twelve hours later than the 1
pures. The process of development in an embryo is greatly
accelerated by the establishment of a circulation, and as a conse-
quence the H pure strain gains markedly upon the H_ hybrids
at this time (compare H5 and hs, Plate III). When, however,
the heart rhythm of the H hybrids is established, it is distinctly
more rapid on the average than that of the HZ pures and this
greater rapidity, other things being equal, initiates more rapid
development. Consequently the speed of development, pre-
viously retarded by the belated establishment of the heart rhythm,
is now accelerated by the introduction of a more rapid heart rate,
and, as a result, all of the healthy H hybrids gain markedly upon
the pure bred embryos and present, after a period of eight or ten
hours, the relative average conditions seen in H6 and hé (Plate
Ill). The hybrid specimens that inherit the least retardation in
the establishment of a heart rhythm and the greatest acceleration
in the rate of heart-beat, actually overtake the best of the H pures
before hatching, a few hatching as early as any of the H pures.
After hatching these same fortunate hybrid specimens outgrow
and outlive the best of the H! pures, and these are the specimens
that show the most marked condition of hyperdominance with
regard to rate of development and resistance to adverse conditions.
The specimens which, on the other hand, have inherited the maxi-
mum retardation in the establishment of a heart rhythm and the
minimum acceleration in the rate of heart-beat are too severely
handicapped to succeed in the race, and they lag far behind, and
become weaklings or monstrosities, carrying the character of ill-
health to an extreme not met with in either of the pure breeds.
This condition might also be considered as a case of hyperdom-
inance.

The case is entirely different for the / hybrids. Here also we

556 Tele Jal Newman

have a blending both in the time of establishment and the rate
of the heart rhythm. The M hybrids acquire a circulation at
least twelve hours earlier than the MW pures, but it is a slower and
less efficient circulation. For a time the M hybrid gains markedly
upon the M pure, but the slower heart rhythm seems to make it
impossible for the circulation to incorporate more than about half
of the yolk and while the W/ pures go ahead and develop into large,
normal young fish the hybrids reach a stage as advanced as
that at which the embryos of other strains hatch, but are left
stranded on a mass of yolk that the metabolic processes are unable
to cope with. The fact that the size of these abnormal embryos
is about equal to that of the H pures on hatching may be simply
a coincidence and not a case of dominance at all.

In the case of the H hybrids the size on hatching is, as was previ-
ously stated, simply a physical necessity since the egg membrane
can contain an embryo of only a definite maximum size.

The darker coloration of many H hybrids may also be explained
as a result of the blending of two characters. These individuals
combine the heavy-bodied type of chromatophore seen in H pure,
the closer aggregation of the latter (also a heteroclitus character),
with the darker pigment of the F. majalis parent. This combina-
tion gives the impression of a decidedly darker coloration. Of
course there are all degrees of blending with regard to’ the shape
and closeness of aggregation of the chromatophores, and in the
depth of color in the pigment, but those individuals that possess
the highest degree of similarity to the parent species in all three
respects give the impression of being more darkly colored than
either of the parent species.

The case of hyperdominance involved in the comparative resist-
ances of hybrid and pure strains to lack of oxygen and the pres-
ence of carbon dioxide, may be explained as the physiological
result of the blending seen in the heart rhythms. The H hybrids,
being endowed with a more rapid and hence more efficient circu-
lation than the Hf pures, are enabled to withstand and throw off
the effects of the accumulating carbon dioxide in the system more
successfully than the H pures. On the other hand the M hybrids,

handicapped by a slower and less efficient circulation than the

The Process of Heredity 557

M pures, and having an equal amount of material to assimilate,
succumb more readily to accumulations of carbon dioxide than do
the / pures.

No physiological explanation is offered for the dominance of
F. heteroclitus in the matter of color pattern at the time of hatching.
This seems to be a case of dominance much like many that have
been described in other forms in connection with definitive char-
acters.

Dominance and Survival

Is dominance the equivalent of survival? This is a question
difficult to answer with assurance. It appears, however, that the
H1 hybrid individuals showing the highest degree of dominance in
characters that are of vital significance, such as time of establish-
ment of heart rhythm and rate of heart-beat, produce at one end
of the series individuals that survive and outlive the best of the
pure bred individuals of either species; and at the other extreme
of the series, individuals that fail to survive, perishing during the
developmental process. Thus dominance of certain good com-
binations of characters means survival, and recessiveness of these
same combinations means early death.

In a sense then the dominants, those with the most nearly
perfect resemblance to one parent in a certain set of vital characters
are the only ones that survive; the recessives, those with the least
perfect resemblance, perish early in the developmental process.
All the survivors show dominance with regard to certain combina-
tions of characters, and, if we were dealing only with end results
we would probably be deceived into considering that we had
a typical case of exclusive inheritance. But we have the advan-
tage of having seen the elimination of the recessives.

We may conclude, then, that the dominance of good combina-
tions of characters is the equivalent of survival, while the recessive-
ness of these same combinations of characters is the equivalent of
failure to survive. In this sense our question “Is dominance the
equivalent of survival ?” is answered in the affirmative.

The dominance of superficial, non-vital characters, such as
color pattern, intensity of pigmentation, etc., may be definitely

558 Jithe Jil Newman

correlated with the dominance of certain good combinations of
vital characters. For example it appears that intensity of pigmen-
tation is, in the H hybrid strain, associated with unusual rapidity of
heart-beat.

Might we suggest the possibility that cases of dominance in
other experimental fields might be explained physiologically in
some such way as has been suggested in the foregoing discussion ?

High Degree of Variability in Hybrid Strains the Result of Vary-
ing Degrees of Compatibility in the Germ Cells of the Two
Species

That hybrids are more variable than pure breeds is a fact long
established and is only emphasized by the data brought out in
this paper. The range of variability is, however, far wider than
might be supposed were only the successful types of hybrids con-
sidered. ‘The following series of types showing the various degrees
of success in development of H hybrids may prove instructive:

1 Many eggs never cleave although fertilized by foreign sperm.

2 A few Beae but break down in the two, four, eight, or six-
teen-cell stages, through some disharmony in the germinal materials
involved. This disharmony is usually noticed in one cell, from
whence it spreads through all the cells of the blastodisc.

3 Many embryos die and disintegrate in advanced blasto-
disc stages.

4 A ccmall percentage of embryos are weaklings, due to some
incompleteness in organization. Many of them fail to establish a
circulation and die at an early age. Others reach a stage similar
to that of an H pure at 48 hours and then die.

5 A large percentage of embryos develop into incomplete and
monstrous forms, such as those shown in Figs. 3 to 8. These show
all grades of incompleteness and discord of parts.

6 A few embryos develop to full size but never hatch unless
released artificially from the egg membrane. Even then they are
sluggish and soon die.

7 A large percentage of embryos are rather pale in color, hatch
rather late, and live only a few days, but do not grow.

oe pe er Se

The Process of Heredity 559

8 About ro per cent of the embryos are larger and more active
than the pure bred individuals, hatch early, some few as early as
the earliest of the pure breeds, are darker in color than the latter,
have a more efficient circulation, outgrow and outlive the best of
the pure strain.

These last individuals are those in which the blending of charac-
ters has produced the most harmonious result.

We see then that there are all degrees of compatibility of ger-
minal material from an entire failure to unite in fertilization to the
production of a type of hybrid that is better fitted to cope with its
environment than either of the pure strains.

The Rhythmic Flux of Characters

Many of the facts brought out by the data presented serve to
emphasize the idea of heredity as a process. Instead of a fixity
of relationship between pure strains and hybrids, there is a constant
flux. At one time we see the paternal influence in the ascendancy,
only to be overshadowed later on in development by the maternal
influence. If we were to make the statement that in M hybrids
the presence of heteroclitus sperm accelerates the process of
development, we would be overlooking the fact that this influence
is at best transitory, that the acceleration is evident only after
about twenty-four hours of development, and that, after a period
of from seven to ten days the M pure strain develops more rapidly
than the M hybrid. The latter gradually loses its developmental
momentum and comes to a complete stop, due probably to its
inability to handle a large mass of yolk that remains unassimilated.

In the case of the H hybrids, too, we would be wrong if we
stated unqualifiedly that the introduction of F. majalis sperm
retards development. In the first place no measurable retarda-
tion is evident until a lapse of about sixteen hours, on the average.
In the second place the more successful of the hybrids overtake
the average H pures after six or seven days, and although the
majority of the hybrids subsequently lose ground, a few maintain
their advantage and after hatching outgrow the best of the pure
strain.

There is no fixity of characters here, but everything is flux and

500 ' H. H. Newman

change. A character then may be conceived of as going through
a series of conditions before reaching the definitive state. It may
have started out as a dominant, become recessive for a time,
dominant again, and so on for a varying number of alternating
phases; and who knows whether the characters that we some-
times call definitive are really the end stages in the process ?
Possibly the further development of the individual as it reaches
maturity or senescence may show a condition of dominance less
dominant or even recessive, while some recessive characters may
come to light that before were unsuspected. If development is
continuous as long as life exists, and we have no reason to doubt
that such is the case, we should be somewhat cautious about our
statements with regard to the permanent dominance of this or
that character. If we limit our statements to some particular
period, such as early maturity or a larval condition, we would
avoid the danger of overstatement.

The Importance of External Factors in Heredity

Experience has taught us that only when we make every effort
to equalize the external conditions of a breeding experiment can
we expect to get anything approaching uniformity of develop-
ment. Slight differences in the physiological condition of the
parents, varying degrees of freshness of eggs or sperm, differences
in temperature and of water content, tend to produce differences
in the developmental process that cannot be attributed to the intro-
duction of foreign sperm. We are then driven to the conclusion
that uniformity of external conditions is as important a factor in
determining a similarity of offspring to parents as is uniformity
of germinal substance.

Heredity is defined simply as a similarity of offspring to parents.
The question arises as to what constitutes this similarity. If the
germ cells from which the offspring develops are similar to those
from which the parent developed, and at the same time the external
conditions of development are similar, there results invariably a
similar developmental process, during which the offspring resem-
bles the parent stage for stage. ‘This similarity of developmental
process, it seems to me, is the essence of heredity. The condition-

The Process of Heredity 563

ing factors of a similarity of process are, first a similarity of ger-
minal substance, and second a similarity of external conditions.
Whether these determining factors are of equal or unequal potency
is a question that scarcely requires an answer. Neither is opera-
tive without the other, and in this sense they are of equal potency.
Any change in either will produce a change in the process of
heredity, and to that extent a dissimilarity between parent and
offspring, which means a failure to accomplish an ideal heredity.
Ideal heredity can never be realized because of the inherent vari-
ability of things. No two organisms ever start out from identical
germ cells, nor do they develop under identical external conditions.
Therefore a striking degree of similarity between parent and
offspring is all that is ever realized.

If then we admit that the essence of heredity is the similarity
of developmental process, we must come to the conclusion that
the study of development and of heredity are identical, except in
that the latter is a comparative study, the object of which is to
determine to what extent the developmental phases of offspring
resemble those of parent, or as in the present work to determine
what relative influence is to be attributed to either parent.

Heredity has long been studied as a static condition instead of
a dynamic process, and it is the hope of the writer that the work
here presented (itself somewhat crude and incomplete) may open
the way to more and better researches in this most interesting

field.

EXPLANATION OF PLATES

Pirate I

Adults of both species of Fundulus used in the experiments. Drawings were made from life by
Miss Ella Weeks of the State Agricultural College of Kansas, and were first published by the writer in a
paper, entitled ‘‘Spawning Behavior and Sexual Dimorphism in Fundulus heteroclitus and Allier
Fish,” which appeared in the Biological Bulletin, vol. xii, no. 5, and was kindly loaned by the editod
for the present use.

The figures are life sized representations of average adults as they appear during the spawning season.
The pronounced sexual dimorphism is well shown. The larger size of F. majalis should also be notde.

Fig. 1. Adult female F. heteroclitus.

Fig. 2. Adults male F. heteroclitus.

Fig. 3. Adult female F. majalis.

Fig. 4. Adult male F. majalis.

THE PROCESS OF HEREDITY PLATE I
H. H. Newman

Fic. 3

Fic. 4
Tue JourNAL or Experimenta ZoOLoGy, vot. v, No. 4

Prate II

Pictorial table, illustrating three stages of the Type Series (Series I).

The top row, H, in this plate and in the two succeeding plates, depicts stages in the development
of pure F. heteroclitus.

The second row, h, represents simultaneous conditions in the hybrid strain F. majalis C and
F. heteroclitus °. ,

The third row, m, simultaneous conditions in the reciprocal cross F. heteroclitus o\ and F. majalis 2 .

The bottom row, M, simultaneous condit:ons in pure F. majalis.

All figures are camera drawings except that the egg membrane has been drawn in w.th the compass.
All figures show a magnification of 12 diameters.

THE PROCESS OF HEREDITY PLATE II
H. H. Newman

TS hrs 24 hrs 4Shrs

Tue Journar or ExperIMENTAL ZoOLoGY, VOL. Vv, No. 4

Prate I

A continuation of the series begun in Plate IJ. The significance of arrangement and labeling of
figures is explained in the legend of Plate II.

THE PROCESS OF HEREDITY PLATE Ul
H. H. Newman

SOh 7aS:

“S54 hrs

Tue Journar or ExreriMENTAL ZOOLOGY, VOL. V, NO. 4

ee sy

Pirate IV

A continuation of the series treated in Plates II and III. Labeling and arrangement of figures -
explained in the legend of Plate IT. ;

THE PROCESS OF HEREDITY PLATE IV
H. H. Newman

VOhrs LIZ hrs TOShrs
19

Tue Journat or ExprrimeNrar ZooLoGy, vou. v, No. 4

Pirate V

Camera drawings of typical specimens of the four strains of Series I on hatching or at a period
equally advanced. 3

H represents a typical specimen of pure bred F. heteroclitus on hatching.

h represents an average specimen of the hybrid strain, F. majalis @ and F-.heteroclitus 2.

m represents a well developed specimen of the hybrid strain F. heretoclitus Gand F. majalis
after 27 days development, after nearly all of the pure F. majalis strain had hatched. The size and
general appearance of this type of hybrid is almost identical with those of the paternal species,
F. heteroclitus, although the embryo developed from a F. majalis egg. The large mass of surplus
yolk, which the embryo is unable to assimilate is seen beneath the head and trunk.

M represents a typical specimen of pure bred F. majalis on hatching.

All figures drawn to the same scale, showing a magnification of 12 diameters.

THE PROCESS OF HEREDITY
H. H. Newman

PLATE V

URS Se
POR ae ay.
Er et
Tt he
Wither
+

Tue JourNnay or ExpeRiMENTAL ZOOLOGY, VOL. V, NO. 4

From the Physiological Laboratory, Washington University Medical School, 1806 Locust Street,
Saint Louis, Missouri.

FURTHER RESULTS OF TRANSPLANTATION OF
OVARIES IN CHICKENS!

¢. CC: GUTHRIE, M.D, PH.D:

With Turee Ficures

INTRODUCTION

Numerous attempts have been made to transplant ovaries in
animals with the view of studying their function after the opera-
tion. In general, such attempts have been unsuccessful.

In 1903 Prof. E. P. Lyon, then at the University of Chicago,
invited me to join him in studying ovarian transplantations in
bitches. Feeling confident that the operations would be unsuc-
cessful with the surgical facilities at our command, and knowing
of the high resistance of fowls to infection and to surgical shock,
I suggested the use of chickens.?

EXPERIMENTS

We operated upon two adult hens but the results were unsatis-
factory. It was in the laying season (February, 1904) and we
attributed the operative results to the unfavorable condition of
the ovaries. We then determined on using young chickens but
before we could carry out this intention Professor Lyon assumed
the direction of another laboratory and was unable to continue
in the work.

During the summer of 1904 (August 18) I exchanged the
ovaries between two black and two white leghorn pullets, weigh-

1A preliminary report was made before the American Physiological Society in Washington City, May
7, 1907. (See Proceedings of the Society, American Journal of Physiology, July, 1907, vol. xix, pp.
xvi-xvii). Results of this work was also reported at the Seventh International Congress of Physiolo-
gists, Heidelberg, August 13-16, 1907. (See Archives Internationales de Physiologie, 1907, vol. v,

p- 108.)
2 Since this paper was written I have learned that Dr. E. P. Lyon, even before my acquaintance with
him, planned a similar series of experiments on pigeons and even made some introductory experiments

on them. His reason for selecting the pigeon was that it has but a single functional ovary.

Tue JourNAL or ExPERIMENTAL ZOOLOGY VOL. ZV, NO. 4

564 C. C. Guthrie

ing about 650 gms. each. One black and one white pullet were
saved for controls. All did well for some time after the opera-
tions but during the winter, before the laying season began, their
condition became extremely poor owing largely to being kept in
inappropriate quarters. They lived until the following Septem-
ber but no eggs were laid, even by the controls. During my
absence after this date, all but one of the hens were lost, including
the controls. The remaining operated hen was killed about
October 15, and preserved.

In size and external appearance the hen that was killed seemed
normal. Macroscopically, the ovary appeared normal in size,
location and in its relations to the surrounding structures. It
was many times larger than when transplanted. At the time
of the operation the Graafian follicles did not exceed I mm. in
diameter while postmortem they ranged from 5 to 10 mm. No
corpora lutea were observed. Histologically, the ovary appeared
normal. ‘The hen was found to be affected with tuberculosis and
the ovary was involved, as evidenced by the discovery of a few
of the bacilli in the stroma.

On August 25, 1906, another series of pullets of the same
strains’ were similarly operated upon, controls being saved as
before. They weighed about 750 gms. each, the white ones
being slightly the heavier. All did well after the operation.
About August 31, they were sent to Columbia, Mo., where they
were kept in a small poultry yard until January 21, 1907, when
they were shipped to this laboratory.t| They have since been
kept in individual pens. The roosters have been kept in small
slightly closed cages except for the time they have been placed in
the pens to tread the hens. Great care has been exercised to
keep the records correct in all respects.

SUMMARY OF RESULTS

No marked differences in egg production were found between
the control and operated hens, nor in the fertility of the eggs.

3 All of the chickens of the single comb black and white leghorns used to date, including the rooster,
have been purchased of E. G. Wyckoff, Ithaca, N. Y.
4 Excepting W3, as per addenda to table.

a ooo

Transplantation of Ovaries in Chickens 565

The eggs® and chicks averaged less in weight from the operated
hens than from the controls.

The operated hens at the beginning of the laying season were
somewhat lighter than the controls.* In other respects no dif-
ferences were observed either in the hens, eggs or chicks.

The eggs became fertile in two to four days after mating. On
cessation of mating the eggs became infertile in eleven to nine-
teen days, the majority becoming so on the fifteenth day.’

Control hens (B, and /,), mated to the rooster of the same
breed gave uniformly black fetuses and chicks in the case of the
black hen, and white fetuses and chicks in the case of the white
hen.

The normal black chicks had grayish-yellow breasts and
throats and frequently the under surface of the tips of the wings
were light colored as well, but the plumage on the entire dorsal
surface was always solid black. ‘The light colored areas on the

5 Table showing weights of eggs

Black hen no. 1 White hen no.1/Black hen no. 2) White hea no. 2/Black hen no. 3) White hen no. 3

(By) gms. | (Wi) gms. | (B2) gms. (We) gms. (Bs) grms. | (Ws) gms.
| ;
58.0 | 60.5 | 43-5 49.0 43-5 | 45-0
62.5 57-5 | 36.0 49.0 45.0 49.0
63.0 58.5 38-5 50.0 51-0 | 46.0
55-5 | 60.0 | 41.5 46.0 48.5 49.0
59-5 61.0 } 4265 49.0 44-5 51-5
62.5 s7-0 | 43-5 52.0 47-5 51-5
Aver, 60.1 59.1 | 40.9 | 49.2 46.6 48.6

Note—The eggs in this table were laid early in March.

6 February 14, 1907, the weights of the chickens were as follows: grams.
Blackshenmovsy (33) erijeeercteere at artes tanta peraeien Selenite itetercleyers Ifislewe Sree 1500
Blacksertiniasczs (9) sretetst=/o’at ars aleee ose oi ays: cf ated cnetare cia) <\cl=) ie =: ofatasstars eustalese enernteva asl arcs 1250
EC MO.GUFD) cco odeocouopa Band Dowtos00h jasgrnonspBunoononogoodposcud 1250
Wlclenes sel Wed) hodonken scansotccuuonahads soccnadoabaananoe auosasoetupdcd 1650
\V ations 6111079) osodepcancna sdcbe ocaondes saoU so coneneasnns saa pbosos AoRe 1480
Wihite hemos 21 (Fig) arareretsrtcratcroreh ole ictal ovene ieteloselese cite oie werelsicies cicie sists bsteisto\eie ei= Gieve 1450
SWihites hen mos3f (i973) layaratelerotelausteyeis'«)=in) fol ctalttelope]el-/olovaleteiaielsta\ele Ill. Not less than 1250
Watonern Cor(G 4d Pe oscadndcedooovsoouLenonogdnagoosogungensasd6su0 sGod0dg00 1990

7 This agrees with results on tnis point published by the Ohio University and Experiment Station,

566 C. C. Guthrie

ventral surface were uniformly black after the first moult. Occa-
sionally a normal black may retain one or several white feathers
in the tip of the wing permanently, but this is of rare occurrence
and such white feathers have not been observed in any other
situation.

The normal white chicks were pure white to light buff when
hatched but after the first moult they were always pure white.

<r
iD

vs
UT

—— es
——SS
+————*t

I8 chicks

Fig. 1 Controls

The black hen (B,) carrying an ovary from a white hen (W,)
mated to the white rooster, gave about equal numbers of white
and spotted fetuses and chicks. (In all cases of very small white
fetuses, spots may have been overlooked.)

The white hen (¥,) carrying an ovary from a black hen (B,)
mated to the white rooster, gave white, black and spotted fetuses
and chicks, ‘The spotted ones outnumbered the others combined.

ees

Transplantation of Ovaries in Chickens 567

The black hen (B,) carrying an ovary from a white hen (WV),
mated to the black rooster, gave ordinary black, and black
fetuses and chicks with white legs, in about equal numbers. In
regard to the chicks from this hen described as ordinary black,
some doubt exists as to whether the ventral light colored area
described for normal black chicks was not lighter and greater
in extent in all cases than in the normal chicks.

The white hen (#¥,) carrying an ovary from a black hen (B,),
mated to the black rooster, gave uniformly spotted chicks, 1. e.,
white chicks with black spots on the dorsal surface of the head,
neck, wings, back or on the tail.

DISCUSSION OF RESULTS®

Color and Markings of Chicks

Owing to the uniform results from the controls (see Fig. 1),
it may be assumed that the strains of chickens used breed true
to color. ‘Therefore any variations in the offspring from the
operated hens were due to other influences.°

The fact that in all cases of the operated hens white or black,
or spotted fetuses or chicks were produced (1. e., the offspring
showed variations from the normal in color markings), shows:

1 That the eggs from each of the operated hens were from the
transplanted ovary. Take hens B, and W, (Fig. 2). These
hens were bred to the roosters of their color. Had some portion
of their own ovary not been removed at the time of the opera-
tion’? (a remote possibility), and was functioning, then we
would have expected solid color offspring like the controls. But

8 Size of hens, eggs, chicks, etc., will not be considered at this time.

° The influence of the operation itself, as well as visual and other maternal impressions, including
telegony and the completeness of the removal of the original ovarian tissue, will be rigidly tested in the
new series of experiments now being started. In the present paper it must be remembered that these
doubtful factors are ignored and the discussion is based entirely upon the results in the six hens herein
reported.

1° Hen B, died as stated in the table, of indigestion. Postmortem examination revealed an ovary in
all respects similar to that of a normal laying hen. This fact alone would seem to be all the evidence
required to meet such objection, for should a minute portion of the ovary be overlooked at the time of

operation, it is hardly to be expected that the Graafian follicles would increase in number.

568 C. C. Guthrie

3chichs

chick

Fig. 2. Showing that transplanted ovaries function

Transplantation of Ovaries in Chickens 569

such was not the case. In the offspring from B,, in which the
male and foster mother were black, black predominated but
white occurred. This must have come through the white ovary.
In the offspring from /¥,, in which the male and foster mother
were white, white was the predominating color but black
occurred. The black therefore must have come through the
black ovary."

If we accept the statement that in ordinary crossing of black
and white breeds, the white is dominant, then we may assume that
the same is not true for this kind of (female) crossing, or that
the original color influence was more strongly preserved in the
black than in the white ovary. From the constancy of the results
in the above two hens, we may conclude that the ovaries trans-
planted into the other two hens, B, and ’,, were the ones func-
tioning during the laying season altel

2 The foster mother exerted an influence on the color of the oh
spring. Take hens B, and ,(Fig. 3). These hens were bred
to the rooster of the opposite color, i. e., of the color of the trans-
planted ovary. Yet in the former the majority, and in the latter
all of the offspring were spotted, 1. e., white with black spots on
the dorsal surfaces. In B,, the male and ovary were white and
the foster mother black; in ,, the male and ovary were black
and the foster mother white. In both cases white predominated
in the offspring. It would seem therefore, if we leave the question
of dominance out of account, that the foster influence of the white
hen was stronger than of the black hen. If on the other hand we
consider the foster influence equal in both cases, then we can
explain the results as due to the dominance of the white in the
male or ovary.

The Character of Feather Markings

In the white offspring, black spots occurred on the back, neck,
head, shoulders, wings and tail in frequency in about the order
given. In size they ranged from a few barbules on one feather
to a patch of feathers the size of a dime. The larger spots were

11 It is interesting to note the difference in the distribution of the ovarian color in these two cases.

570 C. C. Guthrie

observed on the back and head; the smallest on the wings. The
markings of the feathers were not constant even in the same indi-
viduals. Some feathers were entirely black including the shaft,
while others had scattered markings on the vanes involving only
the barbs and barbules. In the black offspring, when white

/2chicks

Fig. 3 Showing influence of foster mother.

occurred it did not appear as a spot but some part of the body,
as the leg was solid white. No white feathers were found on the
backs.

More data must be had on these points before definite conclu-
sions can be drawn. Breeding the offspring may add to our
knowledge along these lines. Similar experiments on chickens

ee ee ee

T rans plantation of Ovaries in Chickens 571

and other fowls and on a number of different species of mam-
mals are now being conducted. Other transmitable characters, as
size, peculiarities of anatomical structure, etc., are being observed.
Also experiments on the transplantation of the testicles are being
made.

It will be of interest to determine if the adult ovaries, or if
more embryonic ovaries will give concordant results when trans-
planted.

It seems that the qualities of the ovaries transplanted in these
experiments may have been modified by the foster mother. Or the
foster mother influence may have only been impressed after fertili-
zation of the egg; or the influence may have been effective both
before and after the egg was discharged from the ovary. A dis-
cussion of possible ways in which such influence might act would
be unprofitable at this time. At present we are justified only in
saying that a field appears to be open to attack with a reasonable
hope of profit.

CONCLUSIONS

1 The ovaries transplanted in these chickens seemed to func-
tion in a normal manner.

2 The color characters of the resulting offspring appeared to
be influenced by the foster mother.

572 C. C. Guthrie

Egg chart and table of results of controls and chickens with transplanted ovaries

Black | White | White White —— Black Black
coe leghorn | leghorn _—ileghorn leghorn leghorn leghorn
— OO
hee Black White | Black leg- | White leg- | Black leg- | White leg-
: leghorn | leghorn | horn, ovary} horn, ovary) horn, ovary | horn, ovary
1907 control | control | from W2 from Bo from W3 from Bz
February Bi Wi Bz We Bs Ws
I |
2 O lost | |
3 | |ONF |
i ONF ONF
5
6 OUNSE
7 ONF
8 ON EF |
9 ONF
10 ONF |
II | ON F
12 |
13 ON F
14 |
15 | ONF
16 | Oss
17 | |
13 | O4/s Bi. F| | OFS
19
20 01/4 Bi. F| OFS | 04/5 Wh.F| 01/2 BL. F
21 | O1/1 Bl. F| | |
22 | | OFS OFS
23 | | O 1/5 Sp.F |
24 OFS | O 1/3 Sp.F| O 1/2 BLF
25 | | O Sp. Ck. O 1/3 BLE
| (Wh. legs)
26 OFS | O1/4WhF| OF S OFS
27 O 4/5 BI.F Os
28 | 02/3 Wh.F O1/6Bl1.F) OS
March |
I OFS | OFS |O1/3Sp.F| 02/3 Sp. F) O FS Os
2 OFS | 0O1/3BI.F OS
(Wh. legs)
3 OFS a O)EtS | OFS Os
4 PeORESy 5 |.0iS O1/5BI.F) 03/4 Sp. F
5 | | OFS
6 os etORS! | O 4/5 Wh.F| 01/3 BI. F| OFS
7 OFS OFS | Os O 4/5 Sp. F

Transplantation of Ovaries in Chickens

S05

Egg chart and table of results of controls and chickens with transplanted ovartes—Continued

FATHER

Black
leghorn

leghorn
contro)

Bi
OFS

O 1/3 BI. F
OFS
O lost
OFS

Os
Os

OBI. Ck
Os

Os
Os
Os
OBI. Ck.

OBI. Ck.

O1/1 BI.F

O Bi. Ck.

| White | White | White | Black Black
leghorn leghoru leghorn | leghorn leghorn
a aml af eco
| White | Black leg- | White leg- | Black leg- | White leg-
leghorn | horn, ovary | horn, ovary| horn, ovary | horn, ovary
control from W, fromB, | fromW, from B3
W, By W, Bs W,
ORS los O1/1 Sp. F
| /os OFS
an OEE:S Os
| OFS
eOcrse | ios Os
Orr's: | Os Os
Os OFS
rr OREISi=0| OFS
| | OFS Os
| OSES) | Os Os
ee OnE: Saas FOsE:S |} OS
PuOEFS.. WOES OFS) =] (Os
| Os |
POIs" hy os (Os
| | Os
Os. ros | | Os
Os | OSs Os
Os | OS |
Os |OS 10S | OS
i os OS | os
| Os Os died Os
Os Os Os
| O Wh. Ck.| 01/4Wh.F |
|
| O Wh. Ck.| OS | OS
| 02/3 Sp.F (OS
O Wh. Ck
O Wh. Ck O Wh. Ck. | OSp. Ck.
O 1/1 Sp.F sick |
O Wh. Ck.| O Wh. Ck. O Sp. Ck.
| O Wh. Ck.| O Wh. Ck. O Sp. Ck.
O 1/1 Wh.F| O Sp. Ck. | O1/1'Sp. F
| 03/4 Wh.F| O 1/2 Sp.F Os

| O Wh. Ck.

| OWh. Ck. |

574 C. C. Guthrie
Egg chart and table of results of controls and chickens with transplanted ovaries—Continued
Black | White | White White Black | Black
grees | leghorn | leghorn | leghorn leghorn leghorn | leghorn
Black White | Blackleg- | White leg- | Black leg- | White leg-
MOpHER leghorn | leghorn | horn, ovary | horn, ovary | horn, ovary | horn, ovary
1907 control control | from W, from B | from Ws, | from Bs
°
April | Bi W, Be ® ||) Guraetielge tare W,
14 | OFS | |
15 |O1/2Wh.F | OS | 04/5 Sp. F
16 | | | | |
17 | | OWh. Ck.
18 }wOeS |
19 OBI Ck. | OWh.Ck. | O1/1 Sp.F O 2/3 Sp. F
20 | O 1/3Wh.F |
21
22.) ..|| OFS OWh. Ck. | O 1/3Wh.F | OSp. Ck.
23) e8| LOVE'S OWh.Ck. | OFS | O Sp. Ck.
24 | OBI. Ck. OFS | O1/3Sp.F
25 | | 0.1/3 Sp.F O 2/3 Sp. F
26 |O1/1 Wh.F
27 Os | Os
28 OFS |
29 (O1/1WhF OS os
30 ls 01S OFS
May
I Os
2 | O1/2Wh.F| OS
3 |
4 | |
5 O 1/1 Wh.F
6 Os Os
7 Os Os
8 Os
9 | O lost
10 | OFS
i Os Oss ||
12 |
3 0 4/5 Wh.F |
143 Yaa
15 | OS i) 2OrS | OS OFS
16 Os | OSs
17 | (Os | Os
18 Os |OFS
19 | |
OFS OFS |

Transplantation of Ovaries in Chickens

575

Egg chart and table of results of controls and chickens with transplanted ovaries—Continued

| Black White White | White Black | Black
sar oe leghorn | leghorn leghorn | leghorn leghorn | leghorn
| | |
Black | White Black leg- | White leg- | Black leg- | White leg-
gers leghorn | leghorn horn, ovary | horn, ovary | horn, ovary | horn, ovary
1907 control | control from W, | from By fromW, | from B;
May Bt | Wi B, | W, B; } Ws
21 | Os
22 Os
23 Os |
24 | Os |
25 |
26 |
27 Os | |
28 Os OFS
29 O 10) |
3° 1e)
31 oO ie)
June |
I
2 |
3 Oo lo |
4
5 |
6 oO oO me) oO
7) \
8 |
9 |
10 | 0 oO
II oO |
12 oO |
13 oO |
15 O soft | 16)
16 O soft
17 (0)
Days Eggs 55 Eggs 52 Eggs 67 Eggs 13 Eggs 20 Eggs 45
135 Fetuses and | Fetuses and | Fetuses and | Fetuses and | Fetuses and | Fetuses and
chicks, 13 chicks, 18 chicks, 20 chicks, 5 chicks, 6 chicks, 12
All black Allwhite | Spotted, 11 White, 3 Black, 4 All spotted
| White, 9 Black,1 | Black with
Spotted, 1 | white legs, 2

Legend: ON F, egg, not fertile; O F S, egg, fertile, spoilt: O S, egg, spoilt, fertility not detected;
4/5, 1/4, 1/1, etc., relative size of fetus, 1/1 indicating full term, i. e., just ready to hatch; BI. F, black

576 C. C. Guthrie

fetus; Wh. F, white fetus; Sp. F, spotted fetus; Wh. legs, white legs; Bl. Ck., black chick; Wh. Ck.,
white chick; Sp. Ck., spotted chick; O, egg, not tested. e

Matings: Beginning February 14 to 17, hens B;, B3 and W3, beginning February 28, were trod
daily by the black Leghorn Rooster (BR) until March 5. From March 5 to March 29 they were not
mated. From March 29 to date, they have been trod daily or every other day as before, i.e., by the
black rooster.

Hens W;, W2 and Be were trod as the above, only the white leghorn rooster (WR) was used.

March 29 hen Bs died from indigestion. Postmortem examination showed her ovary to be in the nor-
mal position and in appearance normal for a laying hen both in size and structure. April 4, hen W2 was
found to be very badly infested with lice. Considerable difficulty was experienced in ridding her of them
and she became emaciated.

Hen Wz met with a serious accident early in January in which she suffered a compound fracture of the
femur from which she recovered only in time to be added to the pens February 25.

The fowls have all been closely confined since February in quarters by no means ideal for the produc-
tion of eggs for hatching.

The failure to hatch fertile eggs is attributed largely however to poor incubation.

From the Zodlogical Laboratory of Johns Hopkins University

HEREDITY, VARIATION AND EVOLUTION IN
PROTOZOA

I THE FATE OF NEW STRUCTURAL CHARACTERS IN PARAME-
CIUM, IN CONNECTION WITH THE PROBLEM OF THE
INHERITANCE OF ACQUIRED CHARACTERS IN UNICELLU-
LAR ORGANISMS

BY
H. S. JENNINGS

Witu 22 Ficures

MELO CUGHIO NS aererereersresie, ciscarcesyelage seni lett vette stete coeinne sceveloree ateu he aroleterarcvocercveercsersrarelehe mucrere ererene oars 577

nies OH yjectiokithe work: ayers) yer taresckayontare tatarctetsiar Talc teystorers Orel tras erayatousteterrtstel starlets oe eistsrte 577

2 General plan of the investigation and principles guidingit.................020eees eee eee 578

ay Placeiof the: present investigationlamithisi plants creel <jajeres elerel-i- eievalelole sprvera.ci stele) etetelsteis)tepa(eie 583

Assumed difference in heredity between unicellular and multicellular animals................+4+- 584

The fate of new structural characters (“acquired characters”). ........20-0.02c2seceeeeceeteees 586

We eeocalizediand unlocalized characterss)asteec cine teeta cise ns incieisievelecietsiecieieraraisisia tetanic 586

2 Typical examples of inheritance and its problems in the Protozoa...............0eeeeeee 586

The fate of new localized structures in Paramecium, with observations on growth and regulation.... 589

We History ofailange mew ap pemda pe tiaras oatay= el=ys)s o al= efa]al=yatei«/<tese/ A la}-\el-\ciasaisieieisieteleteeiatelasiele 589

2 General relations and processes shown in the history of the new structure...............-- 599

3) eihe fatelof other new/stricturesam reproduction ls sjeisi=i<19 ar o1stels =e) elie iste) jaretereicleeiore mere 604

ape PointsyspinesOrappend ages eraterter-jarictersiateleretarleetersisie/-t=at-fetncdars) store avers eee ee Lees 604

bap Antenionend truncate’ ererotessta-,sle eieisiesete mis) steisy= mrelateseleie = eV tered eta cierto steieverersiote 607

ig) Wosterior part of the/bodyitruncate jor Jacking jeje orci 21 eielerjoeletel eis peters ieterstatets ainysier 608

GeAnfermonendsawi thiasproyectin gan ele saraveray-leyieie -taieleoiaie/-taselsteietelsiel-eetyetsieeiei eet sterols 609

¢).Crookedness or/generaliirrepularity,ofiformls,.\cr-raja)e »/<1e/e)nterm jeter ialeieloteisicieieiaio teiatelsve ale 609

tf Behavior of mutilations in LE PLOGUC HOM ayers efor stele hal shatetaretexelncl ately ete tedster eters 614

ApeAcauired| characters thattendjtoibe wherited rapa series wists) )aveis sVeetahelae skele akeisie ett felen stores 618

g Adquirement and inheritance of a tendency for the adults to remain united in chains. . . .618

Eiects ohartificialiselectioncrnyciste eis sie appeemeretin «tniclaim = fale ceisler tajaia a ate tetectetere 620

Effectsiofinatural selections: starcyocciias- ec ecteremateter to otasalareie ec eneyas ole love STS 621

What must be the nature of a new character, that it may be inherited ?.. 0.0.2.0... cece eee eee 622

Examples of modifications from which new inherited characters might result................ 624

Summary and general discussion.......... ibaccitonEeac doant draco sbepsunblcbnocccnucder 625

Waiter purer cltedd iercpeyee tetany edsc-h eters tote hosescustssal evens vel eolstate ln fohvate ehausiAraycene ait dees haseheseteleee CrP cree MG 632
INTRODUCTION

I Object of the Work

The investigations of which the first instalment is here pre-
sented are designed as the beginning of a study of the problems

Tue JourNaL or ExperIMENTAL ZOOLOGY, VOL. V, NO. 4

578 AS: ‘fennings

Of evolution in unicellular organisms. The question in the center
Of interest is: How do new inherited characteristics arise? To
study this question a knowledge of the normal phenomena of vari-
ation and inheritance is required. Our first contributions will
therefore deal with these normal phenomena, with incidental
attacks on the main problem as opportunity presents. We shall
take up inheritance, variation, specific differences, correlation,
growth, regulation, selection, and related topics, dealing with
them by experimental, observational and statistical methods. A
large part of such a study, in the common infusorian Paramecium,
is now complete. The present instalment deals with the definite
and circumscribed problem of the fate of new structural characters.

2 General Plan of the Investigation and Principles Guiding It

In presenting the first instalment of an extensive series of inves-
tigations, it will be well to set forth in an introductory way the
general considerations which have guided the work, together with
its relations to previous investigations by the author. Though
apparently a complete departure from the matters dealt with in
most of my work up to this time, it is in reality a logical continu-
ation of my previous work. ‘The latter has lain hitherto in the
field of the physiology of behavior and reactions. In this field I
have endeavored to analyze and isolate, so far as possible, the
various factors at work, keeping in the foreground of interest the
problem of how the behavior happens to be so largely adaptive.
It is possible to show that certain of the features of behavior—
and precisely certain adaptive features—arise during the life-
time of the individual, by physiological processes which appear
quite intelligible from a thoroughly causal standpoint. ‘These
are the processes known variously as the formation of habits; as
learning; as modification by experience; as expressions of the
readier resolution of physiological states after repetition, etc.

But in this field, as in all other parts of biology, we find many
characteristics, and particularly many adaptive features, which
have not arisen during the lifetime of the individual. Certain
structures, certain processes, certain reactions, often highly adap-

Heredity in Protozoa 579

tive in character, are found to be constituent parts of the organism,
yet they have not arisen in the way mentioned, but are “inherited”
from past generations. Such characteristics, in the field of be-
havior, are spoken of as reflexes, tropisms, instincts, etc.; they
are often of a highly complex character.

Our next task is then to investigate the processes by which these
characteristics have arisen. The problem is parallel—perhaps
rather identical—with that which the student of structure sets
himself when he asks how it happens that the animal possesses
certain complex adaptive structures that are inherited from its
progenitors. We cannot hold that complex characteristics can
arise without any processes leading to them, unless we are pre-
pared to abandon the scientific method Where shall we look for
the processes giving rise to characteristics that do not take origin
in the lifetime of the present individual ?

Clearly, there is but one possibility here. What we call the
“individual life” is not the entire history of this mass of matter
and energy that we call “an animal.’ It has existed for number-
less ages in connection with other individuals, as “‘germ cell,”
or the like. Since the animal becomes modified and adapted in
accordance with certain physiological laws, even in the brief span
of its individual life, it is evident that the unmeasured ages of its
previous existence could hardly pass without the occurrence of
processes of modification. And it is only in this period that the
processes could have occurred which have given it the complex
inherited characteristics that it now has. We have then no alter-
native but to study the nature of these processes, if we wish to
understand the origin of the characteristics under discussion.

Such a study of the processes by which organisms become modi-
fied in the life history of the race is of course as much a part of
physiology as is the study of the processes of metabolism, and it
must be pursued in the same spirit. Most of the existing science
of physiology deals with the rapid processes taking place in the
lifetime of the individual and in its “body.” But of course the
slower processes occurring in the germ material and resulting in
modifications which become apparent in later generations are proc-
esses occurring in space and time, and open to objective experi-

580 IEE Ss ‘fennings

mental investigation, exactly as are other physiological processes.
There is the same reason to suppose them detectible by chemico-
physical means as in the rest of physiology. There is indeed no
reason for making any distinction in principle between these and
the processes of movement or metabolism. The investigator in
this field simply works on a part of the domain of physiology which
has been mainly cultivated independently of the remainder of
that science. In no way is the study of racial processes to be so
much advanced as by considering this field, what it really is, a
constituent part of physiology, and by attacking it from the same
standpoints that have proved their worth in the rest of this science.
Study of essentially this character is well under way in the work of
the modern students of heredity—Bateson, De Vries, Davenport,
Tower, Herbst, and others—though the point of departure has
been in most cases not primarily physiological.

The special methods used—the technique—in a physiological
investigation of racial processes will of course be extremely dif-
ferent from those of an investigation of metabolism or contrac-
tility; it is only in fundamentals that the method of attack must
be the same. Every problem requires its own technique. In
the study of racial processes we have to deal with certain problems
and phenomena which have as a rule not been looked at from a
physiological point of view. They are nevertheless physiological
matters, and need restatement in physiological terms. Let us
attempt this:

Evolution, from this standpoint, is a general name for the physio-
logical processes which result in change of characteristics from
generation to generation. The physiological study of evolution
is the objective and experimental investigation of these processes.

A daptiveness, purposiveness, teleology, etc., are concepts based
on the observed phenomena that the characteristics of organisms
are largely of such a nature as to maintain the processes which we
call life, and thus keep the organisms in existence. From a purely
physiological point of view the teleological problem is essentially
this: How does it happen that combinations of such complexity

Heredity in Protozoa 581

of structure and action can continue to exist ?* Or to put the
question in a way that leads directly toward investigation: What
processes lead to the production of lasting combinations, of such
complexity of structure and action as are found in organisms ?

In considering this question, we are struck by the evident fact
that certain combinations of the various factors making up the
universe are more lasting than others. “Two constituents (as gold
and oxygen) come in contact; they do not unite, and the combina-
tion constituted by their juxtaposition is quickly dissolved by the
incidence of other forces. [wo other constituents (as iron and
oxygen) come into contact; they unite, and the combination result-
ing from their juxtaposition is a relatively lasting one. Such
varying permanence of different combinations is seen in every
field, but it is particularly striking among such complex bodies as
go to make up organisms. Here the persistence of certain com-
binations and the evanescence of others is commonly spoken of as
selection. “The combinations which persist are said to be selected.
The term is undoubtedly, for certain reasons, an unfortunate one.

In the study of organisms, as we have seen, one great class of
problems lies in the question, How can such complex combinations
as organisms be lasting? Now, the study of what combinations
are lasting is precisely the study of so-called selection, and so it
happens that in the investigation of the processes by which organ-
isms have acquired their characteristics, the study of selection
necessarily plays a very large part.

Selection has often been looked at from an extremely narrow
loophole, so that only a small part of it has been seen. In a com-
mon case, only the fact that certain :ndividual animals are more
lasting than others is taken into account; on this selection from
among individuals attempts have been made to base an entire
theory of organic evolution. It would seem incredible that any-
one should suppose the principle of selection to be limited in its
operation to this one class of combinations, did not history show
that such views have been held. Selection is merely a name for
certain aspects of the way the world process takes place. The

*This formulation of the problem we owe essentially to Jensen (’07), whose valuable paper cannot be

too strongly recommended to those who wish to view such problems from a physiological standpoint.

582 HI. S. ‘fennings

greater permanence of certain combinations and activities is evi-
dent everywhere outside the limits of organisms, while within the
system making up the individual organism there are conditions
which require the prevalence of this principle of operation, on a
large scale. To selection, or the greater permanence of certain
combinations, within the organism, we must look for an under-
standing of many of the most important problems of biology, and
particularly of those having to do with adaptation. The study
of the internal adaptations of organisms might indeed be defined
as the search for those combinations of structure and _ activity
that are most lasting. ‘The study of the laws in accordance with
which certain combinations are lasting, while others are fleeting,
must become one of the main lines of investigation. The pioneer
work of Roux (81) in this line was most promising, and has been
followed up to a certain extent; but thorough experimental inyesti-
gations along such lines are what is needed. In the meantime,
the relative permanence of those combinations which we call
individuals must remain one of the chief objects of study. As
Kellogg ((07) has well noticed, we have few, if any, cases even of
this, that are clearly and accurately observed and analyzed.

All together, it is clear that a study of the processes which result
in the complex “adapted” organism must be largely a study of
the relative permanence of different combinations—a study of
selection. ‘This of course requires a study of the chemico-physical
laws in accordance with which the processes are brought about,
and in accordance with which some of their products are more
lasting than others. It is in many respects unfortunate for an
understanding of this line of work that it has received the figura-
tive and anthropomorphic name of selection. When we speak
merely of the relative permanence of different combinations
(whether these combinations are individuals, processes, or chem-
ical compounds), we call up no associations foreign to the matter
in hand, and thus run no risk of arousing misconceptions and
prejudice due to such associations.

In studying the racial processes, that have resulted in giving the
organism its “hereditary”? properties, we meet one great difh-
culty. We cannot reproduce the long series of conditions which

Heredity in Protozoa 583

have acted upon the organism when it lived in connection with
individuals of past generations. We cannot hope, then, to study
the precise processes which have given rise to the particular com-
bination of characteristics which we find in Paramecium or the
dog or in any particular existing organism. All we can hope to do
is to study similar processes in progress, controlling and analyzing
them experimentally, till we work out the laws and principles of
their action. By application of what we thus gain to the results
of processes past, we may hope to reach an rea eresieaa Nats of how
organisms have arisen.

3 Place of the Present Investigations in this Plan

In taking up a study of these racial processes, we must first
learn as accurately as possible what occurs in the passage from
one generation to another; what resemblances and differences are
normally found between members of succeeding generations, and
the like. In other words, we must have a ele of the nor-
mal phenomena of heredity and variation, such as is now being
acquired on a large scale in higher animals. When this is obtained
we may proceed to attempt to modify experimentally the processes
and their results—thus approaching the central problem: How
do inherited modifications arise ?

In such work, the relations found in the simplest organisms
deserve investigation. Here we have reproduction taking place
rapidly (a generation or more a day) and in the simplest forms.
I have therefore undertaken a study of the physiology of racial
processes in the Protozoa. Bearing more or less directly on this
matter we have already a large amount of most valuable work,
such as that by Maupas, Hertwig, Schaudinn, Calkins, Woodruff,
Enriques, and others. I have approached the matter however
from a different standpoint, setting the problems of inheritance
and variation definitely in the center of interest. This results in
somewhat different methods of attacking the subject.

584. H. 8. “fennings

ASSUMED DIFFERENCE IN HEREDITY BETWEEN UNICELLULAR AND

MULTICELLULAR ANIMALS—THE ‘““INHERITANCE OF ACQUIRED
CHARACTERS ”

It is often said, and it seems to be generally assumed, that uni-
cellular animals differ fundamentally from multicellular ones in
heredity.* In the Protozoa there is no separation into cells which
normally die after a certain period (“somatic’”’ or ‘‘body”’ cells),
and cells which continue to live and multiply (“germ”’ cells). The
parent produces progeny by simply dividing, so that parents and
progeny are identical.

This seems to simplify extremely the problem of heredity, or
indeed to remove everything problematical from the subject.
Parents and progeny must be alrke, it is said, because they are
the same. In particular it is commonly held that this removes
from the Protozoa all difficulty as to the “inheritance of acquired
characters’’—characters added during the lifetime of the indi-
vidual and due to environmental action, experience, use, accident,
or the like. Such characters are in multicellular organisms often
called somatic, as distinguished from germinal, and such somatic
characters are commonly held not to be inherited. Where there
is no such distinction between soma and germ, it would seem clear
that there can be no distinction between somatic and germinal
characteristics.

To this difference in heredity between Protozoa and Metazoa
much importance has been attached. If the difference really
exists, the Protozoa are much more plastic in evolution than are
the Metazoa; through the inheritance of the effects of experience,
use and environment, the Protozoa must permit of the ready and
rapid production of varied and adapted types. This point has
been emphasized by many writers. For example, in attempting
to account for the great diversities of organization and action
found among animals Whitman (’99) writes as follows:

“Tn primitive organisms multiplying by simple fission, struc-
tural modifications acquired during the lifetime of the individual

*I use the word “heredity” merely as a brief and convenient term for “the resemblance between

parents and progeny,’ without implying any underlying entity, and without prejudice as to the
grounds of this resemblance.

Heredity in Protozoa 585

would be carried right on from generation to generation, and hence
structural foundations for a whole animal world such as we now
see could be laid in a relatively short period as compared with the
time necessary to advance organization in forms limited to repro-
duction by germs. In fact the fundamentals could all be estab-
lished within the realm of the unicellular Protozoa”’ (p. 307).

In my book on the Behavior of Lower Organisms, I expressed
similar ideas, with particular reference to the inheritance of ways
of behaving:

“Tn the unicellular organisms there seems to be nothing in the
way of this inheritance by the offspring of the reaction-methods
acquired by the parent. ‘There is no distinction between the germ
cells and body cells in these organisms: all acquirements pertain
to the reproductive cells. Through reproduction by division the
offspring are the parents, merely subdivided, and there is no evi-
dent reason why they should not retain the characteristics of the
parents, however these characteristics were attained. If this is
the real state of the case, then in unicellular organisms the life of
the race is a direct continuation of the life of the individuals, and
any acquirements made by the individuals are preserved to the
race” ( Jennings 06, p. 320).

Now, if this difference between unicellular and multicellular
organisms actually exists, it is evidently of the highest interest
and importance. Yet there have been no investigations of the
matter to see if there really is such a difference. Our first task
is then to examine the phenomena from this standpoint; attempt-
ing to determine whether characteristics acquired during the
lifetime of the individual* are inherited by the progeny. At the
same time, we shall keep in mind the broader aspects of our prob-
lem, endeavoring to work out in general the relation of reproduc-
tion in the Protozoa to heredity.

*T use for convenience the term “individual,” as commonly employed, to signify in the Protozoa
the separate free cells. I have no wish thereby to take any stand on Calkins’ contention that the entire
cycle of cells derived from a conjugating pair corresponds to the individual of the Metazoan (Calkins
’06). The present paper deals with certain existing phenomena, which are not altered by the views
one may hold on this point. The relation of conjugation to heredity is to be taken up in a later
communication.

586 H. 8. ‘fennings

THE FATE OF NEW STRUCTURAL CHARACTERS (“ACQUIRED
CHARACTERS ”’)

As we have just seen, it is commonly held that “acquired char-
acters” are inherited in the Protozoa, though not in the Metazoa.
Do experiment and observation show that this is true? Does
the separation of germ and body cells make a fundamental dif-
ference in heredity £

t Localized and Unlocalized Characteristics

In dealing with new or “acquired” characters, it is well to dis-
tinguish two classes. On the one hand are those characters
(mainly structural), that are localized in a definite part of the
body, as cilia, seta, a mouth, etc. On the other hand there are
characters that affect the organism as a whole; such are acclim-
atization or other general modifications due to heat, cold, chemical
agents, etc.; size, method of growth, and the like. The inheritance
of the latter class of characteristics, however acquired, presents
much less apparent difficulty than does the inheritance of the
former. The importance of this distinction between localized and
unlocalized characteristics, in investigations of heredity, has
often been emphasized. Weismann has repeatedly demanded
as proof of the inheritance of somatogenic characters in Metazoa
a demonstration of “the transmission of changes of single definite
parts of the parents to the corresponding parts of the progeny;”’
of the inheritance of “definite parts or localized functions.” It
is clear that a somewhat different problem is involved in the two
classes of cases. We shall take up first localized characters in
the Protozoa.

2 Typical Examples of Inheritance and its Problems in Protozoa

To appreciate the problem of the inheritance of localized char-
acters, we will look at one or two simple cases in the Protozoa;
these will serve to bring the whole problem of inheritance in these
animals to a point.

Paramecium (Fig. 1) has a blunt anterior end and a pointed
posterior end. How does it happen that after fission similar

Heredity in Protozoa 587

features are found in the progeny ? The animal has in the anterior
half an oral groove; near its middle a mouth; near the aboral side
two contractile vacuoles. How does it happen that the progeny
have similar structures? If one of these structures should become
modified in the parent, would this modification appear in the
progeny ?

For a more complex case, we have in Oxytricha (Fig. 2), a
definite, typical distribution of the organs of locomotion. ‘There
are, for example, regularly five large sete in a row near the pos-

Fig.1 Paramecium, to illustrate the problems of inheritance in Protozoa. By mere transverse
fission the blunt, grooved anterior end a would be left with only one individual, the sharp posterior end
(p) with another. m, mouth; g, oral groove.

terior end (S, Fig. 2). In other infusoria, related to this one, these
sete appear in different form, number or arrangement. How
does it happen that after fission the progeny have seta of the same
size, structure, arrangement and position as did the parent? If
the parent loses one of these seta, will the reduced number appear

588 lal sys ‘fennings

in the progeny? Similar questions must be asked for each of
the organs of locomotion and other structures, seen in Fig. 2.

These questions regarding details show that we do not after
all gain much for understanding inheritance in Protozoa by such
statements as that “parent and progeny are the same and so must
be alike.”’ For in simple transverse fission of Paramecium there
is no reason that is at once apparent, why the anterior product
should have at its posterior end a point, as its parent had, nor why
the posterior product should have a blunt anterior end with a

Fig. 2 Oxytricha fallax. Mere transverse fission would leave the five large sete s with only one of
the resulting individuals.

groove along one side; these are not simply passed on, ready made,
to the progeny. Again, the simple transverse fission of Oxytricha
does not account in the least for the fact that the anterior product
of division has the row of five seta at its posterior end. The five
sete might be transmitted directly to the posterior daughter-infus-
orian, but the anterior individual would naturally be left quite
without such structures. Indeed, by repeated mere divisions

Heredity in Protozoa 589

(even if followed by increase in size), progeny would after a time
be produced that would have little resemblance to the parent.

Thus it is evident that even in the Protozoa heredity 1s not a
mere result of subdivision. The question returns with force:
How does it happen that the localized structures of the progeny
are the same as those of the parent? And are they the same in all
cases? Are they the same when the characteristics of the parent
have become changed during its lifetime as an individual ?

We shall take up first the simplest and most marked charac-
teristics—new appendages, spines and the like; marked changes
in the form of parts of the body; all sorts of things that might be
characterized as mutations, abnormalities, monstrosities, etc.
We shall deal at the same time with mutilations.

THE FATE OF NEW LOCALIZED STRUCTURES IN PARAMECIUM, WITH
OBSERVATIONS ON GROWTH AND REGULATION OF FORM IN
THIS INFUSORIAN

By examination of dense cultures of Paramecium* many indi-
viduals were found which differed in certain respects from the
usual form or structure. Some had a short, truncate anterior
end; others a blunt or truncate posterior end in place of the sharp
tip; others were crooked or otherwise modified in form; others
showed angles, teeth or spines on various parts of the body. Many
of these were isolated and allowed to reproduce under observation,
so as to follow the fate of the peculiarity in question.

The method of isolation and culture was essentially that de-
scribed by Calkins (’02). The individuals were placed separately
in the concavities of hollow-ground glass slides, in three or four
drops of hay infusion, which was changed either every day or
every two days. ‘The animals were examined once or twice a day.

I History of a Large New Appendage in Paramecium

I shall first describe in detail a typical case of a new structure;
an individual that bore on its body a spine (Fig. 3).. This case
is particularly instructive because the origin of the peculiarity

*The animals studied had the characteristics usually attributed to Paramecium caudatum. The

question of distinguishing species will be taken up in later parts of this general investigation.

599 Jaleo ‘fennings

was observed, and its history followed for many generations.
The observations on this structure likewise give certain important
results as to the method of growth in Paramecium.

First generation. ‘The ancestor of the race we are to study was
a crooked individual (Fig. 3, a), found in a culture containing
many specimens, where food was getting scarce. I have called
this individual a; we shall use this designation for the race as a
whole, appending certain exponents to indicate the various mem-
bers of the different generations. The anterior individual result-
ing from fission will be designated by the exponent (), the posterior
individual by the exponent (’).

The original individual a was bent just in front of its middle at
practically a right angle (Fig. 3, a). It was isolated at 2.50 p.m.,
May 2, 1907.

Second generation. ‘The first division, during the night of May
2, showed that the crookedness was not to be inherited, though
it had its effects on the progeny. ‘The animal divided transversely,
posterior to the bend in its body. The posterior product (a’)
was normal in all respects, so that it need not concern us further.
The anterior product (a') was of about the form that would be
expected from dividing a behind the bend in its body, save that
the posterior end had become still more irregular. This end was
broad and truncate; nearly triangular when seen from the rear;
it extended backward at two of the angles as two pronounced
points (Fig. 3, °).

Shortly after division the daughter individual a! changed shape
greatly; the posterior end budded out a new structure of nearly
the normal shape for the posterior half of the body (Fig. 3, °).
But this new part formed an angle with the anterior half, so that
the body of this individual was again crooked. At the same time the
anterior end extended a little. The two teeth remained near the
middle of the body, the larger one having been carried back a
little, so that it was a little behind the smaller one.

Third generation. At the next division (forenoon, May 4)
the constriction appeared between the two tooth-like projections,
and the plane of division was oblique (Fig. 3,°). Thus the
smaller one of the two projections was at the posterior end of the

Heredity in Protozoa 591

Z —_s
Fig.3 Transformations in the race a during the first four generations. The anterior end is to the
right. _a, the original crooked individual (first generation). ai, a’, the anterior and posterior products
of its fission. At 3 the individual a! has grown and is dividing, giving the individual 7 (anterior) and
2(posterior). The arrows show the origin and transformations of each individual. For details, see

text.

592 Bb Se fennings

anterior product a™', while the larger projection was at the ante-
rior end of the posterior individual a’. In the period just before
and after the separation of the two parts (which occurred at 10.55)
this larger projection grew rapidly still larger, longer and sharper,
as if it were being pushed out under pressure. Immediately after
division the posterior product a‘? had the form shown in Fig. 3, 4,
the projecting spine being as long as the body was thick, and
situated on the aboral side, nearly at the anterior end.

Now this posterior individual a‘? began to grow rapidly.
Growth was most rapid at the anterior tip; this pushed out so as
to leave the spine at some distance from the anterior end. The
spine itself became still longer and stouter. At the same time
the entire body increased in length, the growth seeming most
rapid at the anterior end and decreasing toward the rear. “Twenty
minutes after division the posterior individual a‘? had the form
shown in Fig. 3, ° :

The change of form now continued much more slowly, so that
at the end of four hours the shape was that shown in Fig. 3, °.

In the anterior individual (a™!) a parallel process of growth
occurred; the anterior part of the body pushed out rapidly, while
the posterior part merely changed shape a certain amount. The
small projection was thus left near the posterior end, on the oral
surface (Fig. 3, 5).

Thus we have now on each of these individuals a definite new
structure, the origin of which we know, while the animals are
quite normal in other respects. The new structures have arisen
during the reproductive processes—at a period comparable, if
there is any such in the life of the infusorian, to the germ cell
period, just before development begins, in a Metazoan. Tower
(06) found that in certain Metazoa changes wrought in the organ-
ism at this stage of its life give rise to permanent inherited modi-
fications, though environmental effects at other stages are not
inherited. We have then perhaps as favorable a case for studying
the transmission of a suddenly produced new structure as we could
expect to find in the Protozoa.

We shall here follow only the history of the large anterior spine,
in a‘? (Fig. 3, °), taking up later the fate of the short tooth in a™'.

Heredity in Protozoa 593

Fourth generation. We left the individual with the long ante-
rior spine in the condition shown in Fig. 3, °. At the next fission
(night of May 4) the spine remained with the anterior product
a**1, while the posterior product a'? was a typical individual
without a spine. In this fourth generation, since the division
had taken place at the middle and there was subsequent outgrowth
of the anterior tip, the spine was left behind the middle of the new
individual (Fig. 3,7). The spine itself had become still longer
and more slender. In structure it was a tube of ectosarc enclos-
ing a narrow canal filled with endosare. It was flexible, bending
readily when it came in contact with obstacles, but it did not
show active movements.

Fifth generation. At the next division (noon, May 6) the plane
of division lay just in front of the base of the spine, so that the
latter went to the posterior individual (a"*"?), and was situated
at its anterior end (Fig. 3,%). The other (anterior) individual
(a) was normal, as usual. In the process of growth, consist-
ing largely in the pushing out of the anterior end, the spine came
to lie farther back than at first, so that in the adult infusorian
it was a little in front of the middle (Fig. 3,°). The spine had
become slightly enlarged at its tip, and bent to the right at about
its middle.

Sixth generation. The plane of the next division (night of May
6) passed just behind the spine; so that the latter was now left on
the anterior specimen, av*'21, while the posterior specimen was
normal. The spine was now bent near the base, so as to extend
backward parallel with the body axis (Fig. 4, °).

Seventh generation. At the next division (night of May 7), the
spine of course went to the posterior individual, a" *"*'? (Fig. 4, 7).
It was situated a trifle in front of the middle of the body. The
spine was now long and curved downward and backward over the
right side of the animal. Its base was much broader than before,
and a shorter spine had pushed out forward from the angle between
the base of the spine and its main body.

Eighth generation. At the next division (night of May 8), the
spine went to the anterior individual (a"*"*"*") and was situated
very nearly at its posterior end, though a little displaced toward

Fig. 4 Diagram of the history of the race a, bearing the spine, for the entire twenty-two generations.

The anterior end and anterior individual are throughout to the right. The numbers at the left indicate
the generations, counting the original crooked specimen as the first. The arrows show the lines of
descent. Only the fission of the individual bearing the spine is followed out in each generation. For
details, see text.

Heredity in Protozoa 595

the aboral side. Its base had become broad and low, extending
between x and y, Fig. 4, 8. There is reason to think that it actually
extended back of x, to the posterior end. It would naturally be
carried back in the backward growth of the posterior tip, but owing
to the abrupt point naturally found here, there is nothing to mark
the end of the base, as there is at y. The anterior point (at y)
had dwindled to a mere knob, while the main spine trailed behind,
half the length of the body.

Ninth generation. At the next division (night of May 9), the
spine passed, as was to be expected, to the posterior individual
qvet21212 Certain interesting changes have taken place in the
position and structure of the spine, which throw light on the proc-
esses of growth, and which have important consequences for
succeeding generations. The free part of the spine is still very
near the posterior end, and stands again at right angles to the body
(Fig. 4,°). The broad base of the appendage (x-y, Fig. 4, °)
has been still farther drawn out in the processes of growth, so
that it extends forward almost to the anterior end (to the point y,
Fig. 4,°). Posteriorly its end is not evident, but it doubtless
reaches to the posterior tip. Thus the base of the spine now
extends nearly the entire length of the body, so that it must be
cut by the next fission plane.

It will be observed that up to this time the spine has regularly
alternated between the anterior and posterior individuals in the
successive generations. This is indicated in the designation
employed (a'*"*"""*""?), the exponent (') indicating in each case the
anterior product of fission, the exponent (*), the posterior product.
When situated on the anterior individual the spine lies back of
the middle of the body (see Fig. 4, °°, etc.) When on the
posterior individual it has always lain in front of the middle of
the body (see Fig. 4, #7), till in the present generation (Fig. 4, °).
These changes in position are due to the growth occurring after
fission; they give us a means of analyzing this growth—a matter
to be taken up later.

Tenth generation. At the next fission (May to, day) the free
portion of the spine went again to the posterior individual, thus
breaking the regular alternation which has prevailed up to this

596 H. S. “fennings

time. The individual bearing the spine is therefore a?"

The effect oe Cs ridge forming originally the base of the sate
(x-y, Fig. 4, §) is shown in the fact that the two individuals did
not se as usually happens; they remained connected by a
sort of bridge passing along the aboral surface (Fig. 4, 1°). Evi-
dently the eubsence formed by the extended base ofthe spine 1s
not so easily cut by the processes of fission as are the other parts
of the body; it therefore forms the bridge. The two individuals
thus connected did not move in unison; there was much pulling,
bending and twisting of the slender connecting bridge, so that the
latter appeared likely to break. In the course of time this hap-
pened; the two individuals separated some time during the next
night, before the next fission occurred.

As will appear in the sequel, this tendency to remain connected
even after the adult condition is reached persisted in the progeny
of these individuals for many generations. We have therefore
something resembling the inheritance of a new characteristic.
This matter will be taken up in a separate section.

The spine still remained near the posterior end of the individual,
though not so near it as in the previous generation. ‘The posterior
tip has pushed backward from the spine, in the growth that takes
place after division. It carries with it some portion of the base
of the spine, just as happens in front.

Eleventh generation. Again the spine went to the posterior
individual (night of May 10). As would be expected, the spine
is now further forward; it is again nearly straight and at right
angles to the body (Fig. 4, '').

Twelfth and iisneenth ge nerations. During the night of May
11 there were two fesions. giving three specimens of the normal
form, and one with the spine. It appears clear that at the first
of these two divisions the plane of fission was just in front of the
spine, so that the latter was left almost squarely on the anterior
up of the posterior individual; here it remained till the next divi-
sion. ‘This time of course the spine went to the anterior indi-
vidual, still remaining almost exactly at the anterior end. In its
outgrowth the anterior tip has carried the spine with it, owing to
the fact that the latter was almost at the very end. | The individual

Heredity in Protozoa 597

bearing the spine in the thirteenth generation is therefore to be
designated QU PN 21.21-2.2-2-201

Fourteenth and fifteenth generations. During the night of May
12 there were again two divisions, giving three normal individuals
and one with the spine. The spine is now situated at about the
middle of the body (Fig. 4, %). The only way this result can have
been reached is as follows: The spine went to the anterior indi-
vidual in both of these divisions, and in the growth processes after
each division it moved backward about one-fourth the length of the
body (or rather, the anterior tip grew forward thatamount). The
individual of the fifteenth generation is therefore at?t?1242222414,

The spine now bears a ball at its tip (Fig. 4, °). This is due to
the fact that at the time of fission some of the endosarc is squeezed
out through the tube of ectosarc, thus forming the ball. This
indicates that at the time of fission, or in the period of rapid
growth just following it, the internal contents must be under
much pressure.

Sixteenth generation. The plane of fission (night of May 13)
passed just in front of the base of the spine, leaving the latter at
the anterior tip of the posterior individual (Fig. 4, *). Again
it failed to be displaced backward in the growth following fission.
The ball at the end of the appendage was gradually constricted off
from the tip, becoming completely separated at 10.15, May 14.

Seventeenth and eighteenth generations. During the night of
May 14 the animal again divided twice. The method of division
is shown clearly by the fact that the three individuals without the
spine remained connected in a chain, only the animal bearing the
spine being free. The spine went to the anterior individual in
both fissions, being displaced backward about one-fourth the
body length in each growth period.

Nineteenth generation. In the next division (night of May 15),
the spine went to the posterior individual, being borne again at the
anterior tip (Fig. 4, 1°).

Twentieth and twenty-first generations. During the night of
May 16 there were two generations, the spine going to the anterior
individual in each case. This is demonstrated by the fact that the
three individuals without the spine have remained united in a chain

598 dale AY Fennings

while the spined animal is free. The spine 1s still at the anterior
tip; it has not moved backward for two generations.

This individual did not divide for more than twenty-four hours,
and during its lifetime the spine became a little shorter. The
animal now used the spine almost continually. It placed the tip
of the spine against the bottom of the vessel or against any other
surface, then ran along the surface, keeping the tip of the spine
in contact, while currents of water passed down the oral groove
(Fig. 5). This use of the spine is of course incidental to the com-
mon habit of these animals, of placing one side of the body against
a surface and running along it. But this is the first generation in

See ee
ae

Fig. 5 Use of the spine by the individual of the twenty-first generation. The tip of the spine is
pressed against a surface and the animal runs along it, in the direction indicated by the large internal

arrow, while the currents of water down the oral groove to the mouth are indicated by the small arrows.

which such a use of the appendage occurred. This, taken with
the fact that the appendage seemed to be gaining a permanent
position at the anterior tip suggested possible interesting develop-
ments in the future.

Twenty-second generation. The spine again remained at the
anterior tip. The division (afternoon of May 18) was at first not
complete (Fig. 4, *), the animals remaining connected for more
than twenty-four hours.

On the morning of May 20, the two had separated, but had not
divided farther. Both were swollen and opaque; they were evi-

Heredity in Protozoa 599

dently in an unhealthy condition. Investigation showed that the
wrong sort of bacteria had multiplied in the culture fluid last
made, making it opaque and sirupy. All the specimens (for
various other experiments) that had been placed in this fluid were
unhealthy or dying. This multiplication of injurious bacteria
in culture fluid made in the usual way, is a not uncommon and
most disastrous occurrence. To it we shall return in another
connection.

The two sister individuals (one with the spine, the other without)
were transferred to clean water, and later to new culture fluid.
They were still living May 21, three days after the last preceding
fission. But on the morning of May 22 I found, to my great
regret, that the individual with the spine had died. Its sister
recovered and propagated the race for many generations, of which
we shall have to speak in our account of the hereditary tendency
to remain connected after fission.

The last individual bearing the spine was designated
Qe eatin “These‘exponents show to which) mdivid=
ual the spine passed at each division—(') indicating the anterior
individual, (*) the posterior one. ‘The spine was traced through
twenty-one generations (the first generation not having the spine).
Fig. 4 gives a diagram of the entire history of this structure.

In this history of a localized new structure for twenty-one gen-
erations, certain general relations appear, which we will here set
forth, though a full discussion of their significance will be reserved
till other cases have been considered.

2 General Relations and Processes Shown in the History of the
New Structure

1 The new structure was transmitted in each generation to but
one individual. ‘Thus, in the sixth generation there were thirty-
two individuals, with but one bearing the spine (see Fig. 6); in the
eleventh generation, out of 1024 individuals, but one had the spine;
in the twenty-second generation the spine was found on but one
individual out of 2,097,152.

Furthermore, the spine occupied a definite place in the series
of individuals produced. As we have seen, and shall see farther,

600 Ee S. Fennings

sometimes Paramecia do not completely separate after division,
but remain united in chains. If we conceive of all the individuals
of each generation as thus forming a chain, each being in the posi-
tion that the method of transverse fission gives it, then on such

Fig. 6 Diagram showing the position and relations of the spine of a in the sixth generation, when

thirty-two individuals are present. The individuals are conceived to have remained united, in the posi-
tions given them by the successive fissions. The spine would be found on the eleventh individual from
the anterior end of the chain (drawn with a heavy outline).

a chain we would find but one spine, having a certain definite posi-
tion. ‘Thus, in the sixth generation, where thirty-two individuals
were present, the spine would have been situated as shown in
Fig. 6, on the eleventh individual from the anterior end of the

Heredity in Protozoa 601

series. In the ninth generation, after eight regular alternations
from the anterior individual to the posterior one and back, in the
fissions, we should have a chain of 256 individuals, with the spine
on the 171st individual counting from the posterior end of the
chain. In the twenty-second (final) generation, the chain would
be 2,097,152 individuals long, and would bear but a single spine
situated on the individual numbered 1,393,592 from the posterior
end.* Such a chain would be about 419 meters long, with the spine
about 278 meters from the posterior end.+

Thus though the new structure is transmitted it is not multi-
plied, and there is no tendency to produce a race with this char-
acteristic. [here is evidently a fundamental difference between
on the one hand this simple handing on of a localized structure
to one of the new individuals, and on the other hand, the reappear-
ance of the localized structure in all or many of the individuals
resulting from fission. ‘The difference is in some respects similar
to that between “somatic” and “germinal” characters in Metazoa.
This point we shall take up later.

2 The position of such a structure on the body of the individual
is not permanent and the same in succeeding generations. The
same structure is found in one generation at the anterior end, in
another at the posterior end; now at the middle; now in some
intermediate position. At first the structure alternated regularly
between a position nearer the posterior end, and one nearer the
anterior end; later its wanderings were wider.

These fluctuations of position are due mainly to the processes of
growth following fission. These processes will be analyzed quan-
titatively in later communications; here we see merely the main
facts in a general way. After fission the entire body lengthens,
both ends pushing out rapidly. The anterior tip pushes out
somewhat more than the posterior one. In consequence, a struc-
ture located, just after fission, near the anterior end (Fig. 3, §) is

*The rule for finding which individual of a given generation would bear the appendage is as follows:
If in a certain generation the number of individuals posterior to the one bearing the spine is x, then in
the next generation, if the spine goes to the posterior product the number posterior to the spined individ-
ual will be 2x; if the spine goes to the anterior product, the number will be 2x + 1.

+The length of a single individual being taken as 20044.

602 H. 8. “fennings

left behind in the growth of the tip of the body, so that in the adult
infusorian it lies halfway back to the middle of the animal (Fig. 3, °).
At the next fission it of course goes to the anterior product, lying
at or behind its middle. By the greater growth of the anterior
end it is further displaced backward, so as to lie clearly behind the
middle. At the next fission it must then go to the posterior prod-
uct, and be near its anterior end. Now it is again displaced
slowly backward, the same processes being repeated. Thus the
process is normally one of steady movement backward, interrupted
by fissions which at intervals leave the spine near the anterior
end of the posterior individual. A diagram showing this normal
course of events is given in Fig. 7.

Sometimes through irregularities in growth, or other cause, the
structure comes to be situated very near to or at one end (as in
Fig. 4, °""°**). Then the course of events becomes. slightly
different. Ifthe structure is near the posterior end (Fig. 4, *) the
posterior tip grows back from it only a little, so that it still remains
behind the middle of the body. At the next fission it therefore goes
to the posterior individual (as it would in the “normal”? course).
Now the posterior end again grows back but a little, while the
anterior tip grows much, so that the spine is still behind the middle.
It therefore goes again to the posterior individual. It may thus re-
quire as many as three fissions to bring the structure to the middle,
so that it passes again to the anterior individual, reéstablishing
the alternations (Fig. 4, °°”).

Is situated at or very near the anterior tip, the structure is car-
ried forward in the growth processes; it may therefore remain for
several generations in this region (Fig. 4, *°'°**), before it is dis-
placed backward sufficiently to lie behind the middle. Possibly
a structure might in the course of time attain a permanent posi-
tion at the anterior tip. This seems indicated by the last three
generations of a.

Thus on the whole the general tendency of the growth processes
is to shift any surface structure from the ends toward the middle
of the body, while the fissions again transfer it toward one end;
with the further result of an alternation of position from the
anterior to the posterior product of fission and back again.

Heredity in Protozoa 603
In general then it must be realized that the parts of the body
of the infusorian do not have a permanent definite relation to the

form or structure. A portion of substance that is anterior in one
generation is posterior or median in another. Thus definite

Ve aS

Ww
&

a ks

4 a

Fig. 7 Diagram showing the usual regular changes in form and alternations in position of the spine
through four generations. The numbers at the left indicate the generations, a younger and an older
stage being shown in each generation (save the fourth). a is the anterior product of fission, p the pos-
terior one. In generations 1 and 3 the spine is on the anterior half of the posterior daughter cell; in
generations 2 and 4 it is on the posterior half of the anterior daughter cell.

604 H. S. fennings

pieces of substance are not necessarily permanentiy differentiated
to play certain parts. ‘The organism 1s plastic, and is made over
at fission. The normal reproduction involves the same work-
ing over and re-differentiation—‘‘morphallaxis”—that occurs in
regeneration.

3. Yet this making over is not complete. Oral and aboral
surfaces retained their relative position throughout these twenty-
two generations, the spine remaining always on the aboral sur-
face. Furthermore, the entire history shows that a given struc-
ture may be bodily transmitted for many generations without becom-
ing greatly changed. It may even, finally, acquire a more or less
permanent position, remaining for at least several generations.

In the normal reproduction we find structures which behave in
both of these ways—some being directly transmitted, others
re-made. The two contractile vacuoles of Paramecium pass
bodily, one to each of the progeny—though each individual forms
likewise one new one. ‘The mouth and pharynx are said to pass
to the anterior product of fission, the posterior product forming
new ones. ‘The oral groove, the blunt anterior and the pointed
posterior end, these are examples of structures that disappear in
reproduction and are made anew. ‘The cilia and seta of the Hypo-
tricha are not transmitted, but produced anew in the new individ-
uals. Fission is on the whole mainly a process of reorganization
and new production, rather than of transmission.

3. Fate of Other New Structures in Reproduction

The fate of many other new structural peculiarities was fol-
lowed in various individual.lines; after the detailed account we
have given above, these can be set forth briefly.

a Spines, Points or Appendages

In many cases studied the history of points or appendages on
the body differed from what we have described above for the line a.
1 This is the case with the small point on a‘, already mentioned.
(Fig. 4,%). As will be recalled, there resulted from the division
of a’ two individuals bearing spines or points; we have followed

Heredity in Protozoa 605

the history of the large spine of a'* and its descendants. We will
now follow briefly that of the short posterior spine of a’! (Fig.8,").

The next division (night of May 7) was of course at about the
middle of the body, so that the anterior product a‘ was a normal
individual without a spine. The posterior product a‘** had the
spine in about the same position as in the previous generation,
though it shifted during growth a little farther forward (Fig. 8, ”).

At the next (fourth) division the point passed to the posterior
product (a'!**) and remained in nearly the same position as before
(Fig. 8,%). It had become smaller, so that it was now a mere
lump, hardly noticeable.

/
CA

Pee ea
=
2 el ag aie

Fig.8 Diagram of the history of the small tooth in the race a. Sce text.

At the next fission (fifth) the point or lump quite disappeared,
being in some way reduced during the growth processes accom-
panying division. Both individuals resulting from fission were
of the normal form (Fig. 8, *).

Thus this small posterior point persisted through but three gen-
erations, and in each generation it was found in butone individual.
A process of regulation of form took place slowly, accompanying
the changes involved in fission, till finally the new structure had
disappeared.

606 H. 8S. Ffennings

2 Inaline or race which I called am, the course of events was
as follows: ‘The ancestor am was a short individual, seeming to
lack almost completely the posterior half of the body. In the
first two fissions the anterior product was in each case a normal
individual, while the posterior product was more or less abnor-
mal, with a blunt irregular posterior end. In the fourth genera-

Fig.9 A number of generations in the history of the race am, showing the shifting, transformations,
and gradual disappearance of the spine. The numbers at the left indicate the generations figured. The

spine first appeared in generation 4 and disappeared in generation 11.

tion there were two abnormal individuals, one of which bore a
short spine projecting from its aboral surface, at about the middle
of the posterior half of the body (Fig. 9, *).

In the fifth generation the anterior individual was normal, while
the posterior one bore the spine a little farther forward than in

Heredity in Protozoa 607

the previous generation. The spine itself was a little longer (Fig.
9°).

In the sixth generation it still further increased in length at
the time of division, and went again to the posterior individual
(Bie295-°):

In the next two divisions the tooth went in each case to the pos-
terior product, and continued to grow smaller. It remained near
the posterior end, and in the tenth generation (am??*??1???) it
was hardly noticeable (Fig. 9, ‘°). During the next division it

Fig. 10 History of a race derived from an individual with a truncate anterior end. The numbers
indicate the generations figured. The truncate end is barely visible in generation 3, but had quite dis-

appeared in generation 4.

disappeared completely, both products being typical individuals
(Eig. 9; 7).

Thus this spine persisted through seven generations, first in-
creasing in size, then decreasing, till it disappeared.

b Anterior End Truncate
In three cases I followed the history of the progeny of individ-

uals having the anterior end short and sharply truncate, as if cut

off by a knife (Fig. 10).

608 ae S. ‘fennings

In each case the truncation of the anterior end persisted for a
few generations (two to five), being transmitted of course to but
one individual in each generation. At each fission, as a rule, the
peculiarity of the anterior end of this individual became less
marked, till it became invisible. There is thus a marked tend-
ency at the time of division to regulate the body form, bringing
it back to the normal condition.

c Posterior Part of the Body Truncate or Lacking

Many individuals were found in which the posterior half of the
body seemed almost lacking. The body ended bluntly just

Pe
pOttrS Fae

Fig. 11 History of a race derived from an individual in which the posterior part of the body was
extremely short and rounded. The posterior end is to the left. The peculiarity was transmitted to one

individual in each generation, becoming less and less marked, till in generation 5 it has disappeared.

behind the mouth. The animals were about half the normal
size, and presented much the appearance that would result if they
had been cut in two transversely just behind the mouth (Fig. 11).

I followed the history of ten cases of this sort. In all cases the
bluntness of the posterior end is transmitted, usually in weak-
ened form, to the posterior individual resulting from division,
while the anterior individual is quite normal in form. This con-
tinues as a rule for three or four generations, the posterior end
approaching after each division more nearly the normal form,
ull finally regulation is complete, and all the progeny have the
usual shape. A typical case is shown in Fig. 11.

Heredity in Protozoa 609

In one case the sharply truncate form of the posterior end was
transmitted almost unchanged to the posterior progeny of the first
divisions, though the posterior half of the progeny was much longer
than in the parent. But in three more generations the posterior
individual, like all the others, had reached the normal form.

d Anterior End with a Projecting Angle

In a certain culture there occurred a number of individuals in
which the angle at the right of the anterior end was in a marked
degree longer than others. These Paramecia ran over the bot-
tom with the oblique surface of the anterior tip against the solid,
suggesting that the projecting angle was due to this action. The
angle disappeared in the changes connected with fission and did
not reappear in the progeny.

e Crookedness or General Irregularity of Form

A considerable number of cases were studied in which the body
of the progenitor was crooked, or was otherwise irregular in varied
ways.

Such irregularities do not pass as such to the progeny. They
usually cause modifications in some or all of the progeny for sev-
eral generations, but these modifications are not repetitions of the
parent forms. They result from abnormalities in fission due to
the irregular form of the parent. Four categories of cases may be
distinguished: (1) Those in which the irregularity of the ancestor
induces in certain of the progeny various peculiarities that con-
tinue indefinitely; (2) those in which complete regulation finally
occurs, all the individuals returning, after a number of generations
to the normal form; (3) cases in which the result is to cause, in
some or all of the progeny, still greater irregularities, resulting
finally in monstrosities which cannot perform the vital functions
properly, and therefore die; (4) cases in which the irregular indi-
viduals do not reproduce at all; they persist for a time, and finally
die. Typical cases of each of these categories may be described.

1 The individual a, whose history has already been followed
(pp- 589-604), is anexample of the first category. Here the crooked-

610 Hele S. fennings

ness of the parent (Fig. 4, !) caused a spine to appear on one of the
progeny; this persisted on a single member of each generation, as
long as it was followed (22 generations). The other progeny were
normal.

2 The individual a/ was bent a little in front of the middle so as
to form nearly a right angle (Fig. 12,1). At the first division
the posterior product was of the normal form, while the anterior
product was somewhat irregular (Fig. 12, *) but less so than the
parent. When this divided, the two individuals resulting were
both of the normal form. Regulation occurs during the process
of fission.

EN 12>
aa

Fig. 12 History of a race derived from a crooked specimen. The crookedness had disappeared in
the third generation.

The individual ab had the posterior end crooked (Fig. 13).
When this animal was placed in the culture fluid, it became
plumper, and the abnormality of form was less marked (”).
When it divided the anterior product was of the normal form,
while the posterior product had the posterior point slightly dis-
placed toward the aboral side, but was otherwise normal (Fig. 1332)
When it again divided, its progeny were both normal in form.

The case of aj Belones partly in the second category, partly in
the third. The body of the parent aj was small and irregular,
with a broad anterior end, which bore on one angle a projecting
point (Fig. 14, +).

When this was placed in the culture fluid it did not divide for
three days. [he body increased in size and especially in thick-

Heredity in Protozoa 611

ness, and the projecting angle became more marked (Fig. 14, ").
On the third day it divided; the posterior product was normal in
shape, though smaller than usual, while the anterior product
was extremely irregular, having the form shown in Fig. 14, *.

In the next twenty-four hours this irregular structure underwent
a partial division, increasing its size and irregularity of form (Fig.
14,°). The structure thus produced was double, since it had two
mouths (77), both of which took food; and there were two independ-
ent protoplasmic circuits for the digestion of food.

During the next twenty-four hours this structure divided into
two very unequal parts. One product was a short, somewhat

Fig. 13 History of a race derived from an individual with a crooked posterior tip. The irregularity
had nearly disappeared in the second generation; in the third (not shown) it was quite gone.

irregular individual. ‘The larger product was still very irregular;
it represents three united individuals (Fig. 14, ‘).

The smaller product divided again, producing progeny that were
normal in form, though small in size.

The larger product, composed of three incompletely separated
individuals, did not divide again; after two days it disintegrated.

3. The individual aq represents mainly the third category, in
which the irregularity of form is increased in reproduction, till
death occurs. This specimen was curved as shown in Fig. 15, a.
At its first division the products did not completely separate, but
formed the structure shown in Fig. 15,5. At the next division
the right half divided in such a way as to produce one nearly nor-

612 H. S. ‘fennings

mal free individual, while the other product remained attached
to the left half. The latter underwent a partial, irregular division.
Thus the result is to produce an irregular structure consisting of

three fused individuals (Fig. 15, c).

Fig. 14 Diagram of the history of the race derived from the irregular individual aj. In the third and
fourth generations double and triple monsters appeared, with several mouths (m) and multiple proto-
plasmic circuits. Two such circuits are shown by arrows at 3.

Heredity in Protozoa 613

This structure underwent other partial fissions, giving the irreg-
ular monster shown in Fig. 15, d. This lived for about four days,

then disintegrated.
4 Instances of the fourth category, in which no divisions

occurred, are given by aq' (Fig. 16,4) and am??? (Fig. 16, db).

Fig. 15 History of the race derived from the irregular individual aq. See text.

These both lived for five days without dividing or taking food;
both then disintegrated.

The mass ar was the result of partial fission, so that it included
several partial individuals. As successive partial fissions occurred

614 TTS; ‘fennings

it took various forms, of which the three given in Fig. 16, c, d, e
are types. This structure took food by five or six mouths, and had
a number of partly independent systems of circulation. It reached
a length of 450, with a breadth of 150”. The normal Paramecia
in the same culture in which it occurred showed dimensions of
about 1504 x 60. This structure had therefore the bulk of
about twenty normal individuals.

This was kept for ten days, but finally it disintegrated.

Fig. 16 Irregular individuals which do not divide farther. a and b are separate individuals that
finally died. c,d and ¢ are stages taken at intervals of several days in the complex mass ar. _m, mouths.

f Behavior of Mutilations in Reproduction

Paramecium differs from many of the infusoria in the fact that
it does not stand mutilation well. The internal contents seem
very fluid, so that they flow out as soon as the ectosare is cut; the
animal at once disintegrates. It is therefore difficult to study
the regulation of injuries in this animal, either during the active
life, or at reproduction.

However, from a large number of experiments, certain results
were reached that show how mutilations behave, both in ordi-
nary regulation and in reproduction.

Heredity in Protozoa 615

1 Mutilations in adults. Whenever the ectosarc is punctured,
the internal contents flow out and the animal dies. But in a few
cases mutilations were produced without puncturing the ectosare.

Thus, a fine glass rod was drawn across an individual near its
middle; leaving a deep constriction, while the two halves of the
body were swollen (Fig. 17, a). “Fhis constriction persisted for
some hours, becoming gradually less marked. The next day the
animal was perfectly normal.

In another similar experiment, blister-like swellings were pro-
duced, and the anterior portion of the body became totally irregu-
lar (Fig. 17,6). But within 24 hours the normal form was com-
pletely restored.

Thus it is clear that the adult Paramecium has the same power
of regulating form that is so well known in Stentor and other infu-

Fig. 17 Mutilations produced by drawing the tip of a glass rod across the adult animal. See text.

soria. But this can come into play only when the injury has not
been of a nature to puncture the ectosarc and so to cause disinte-
gration.

Many attempts were made to remove only a part of the internal
fluid (endosarc), without causing death. The ectosare was
pierced with the tip of an excessively fine capillary glass rod.*
But in all cases where any of the endosarc flowed out, the remain-
der followed, and the animal died.

2 Mutilations in dividing specimens. It was thought pos-
sible that specimens undergoing fission might show a different
physical state of the protoplasm, such as to permit mutilations
without immediate disintegration. To a limited extent this was

*These can easily be made so fine that the tip is apparently not larger than a cilium of Parameciume

616 lake ss fennings

found by experiment to be true. When a specimen undergoing
fission is pierced with the tip of the glass rod or otherwise muti-
lated, it does not go to pieces so rapidly as the adult, though in
most cases it finally disintegrates. But in a few instances speci-
mens thus treated survived.

Thus, while the Paramecium ma was undergoing fission, its
anterior half ma’ was pierced with the rod, allowing a part of the
internal contents to escape. This half became distorted (Fig.
18, a) while the other half became swollen. The latter resumed
later its normal form, and fission continued. The injured half
a retained its distorted form (Fig. 18, 4). During growth the form
became somewhat nearer normal (Fig. 18, c), but complete regu-
lation did not take place in this generation.

Fig. 18 History of the specimen ma, mutilated during fission. See text.

During the night the irregular individual divided. ‘The anterior
product was quite normal in shape; the posterior one still showed
a slight irregularity of form at the posterior end. At the next
fission this disappeared and both products were normal.

Thus the effects of the mutilation persisted in some of the indi-
viduals for three generations, then disappeared.

In a number of other cases young or dividing specimens were
marked with deep furrows by pressing them with the rod. These
marks lasted some hours, but disappeared before the next fission
occurred.

In the dividing specimen mb the posterior part mb? was
pierced with the glass rod, so that a part of its contents escaped,
while by contraction most of the remainder of its contents were
forced into the anterior half mb‘ (see Fig. 19, 6). Thus the

Heredity in Protozoa 617

pierced part became very small; later it increased in size and
became irregular (Fig. 19, c). The fission was never completed,
this irregular part remaining attached to the posterior end of
the normal individual mb.

The normal part mb' divided twice, budding off, as it were, two
normal individuals at its anterior end; its posterior part remained
with the irregular mass attached, as in Fig. 19, d.

At the next division the two components remained connected,
with the irregular mass attached to the posterior end (Fig. 19, e).

ee ere

e) O

”

Fig. 19 Effect of mutilation during fission in the specimen mb. See text.

The irregular mass had itself made some attempts at fission, with
the result that it became still more irregular.

There was no further change for three days; then another partial
fission produced the results shown in Fig. 19, f.

During the next day the entire structure disintegrated. In this
case the effects of the mutilation lasted for several generations,
finally causing death.

All together, it is clear that while mutilations may be passed on
bodily to certain of the products of division for a number of gener-
ations, there is no tendency for them to be inherited by all the

618 Tel eek ‘Fennings

progeny; no tendency for the mutilation to be duplicated in new
individuals. There is no tendency to produce a race of mutilated
individuals, any more than there is in Metazoa. Regulation takes
place at the time of fission, so that after several fissions the normal
condition is restored.

4 Acquired Characters That Tend to be Inherited

g Acquirement and Inheritance of a Tendency for the Adults to
Remain United in Chains

The acquired characteristics thus far described have shown no
tendency to be inherited in such a way as to produce a race bearing
the new character in question. We now come to a case in which
such a tendency actually showed itself. The difference between
this case and the others is instructive, suggesting what must be the
essential nature of an acquired character that may be inherited.

The characteristic in question is a tendency for the adult indi-
viduals to remain united in chains. This tendency appeared in
the line a, which we have already described in connection with.
the transmission of a long spine (pp. 589-604); the beginnings of
the characteristic now under consideration have been set forth in
that description. In the process of growth the broad base of the
long spine (Fig. 4,7) became drawn out, till in the individual
a'?124242 it formed a ridge running along the aboral surface
nearly the entire length of the body (Fig. 4, °). At the next fission
it was found that the fission plane did not pass so readily through
this ridge as through the remainder of the body, so that the two
resulting individuals did not separate, but remained connected
by a bridge passing from the aboral surface of one to that of the
other (Fig. 4, 27°).

The continued union of the two individuals after fission reap-
peared in succeeding generations, both in the individuals formed
from the region anterior to the spine (as in Fig. 4, 1°), and in those
formed from the region posterior to the spine (Fig. 4,17 7°). In
the eighteenth and twenty-first generations three individuals
formed a chain (Fig. 20, a). In succeeding generations many
such connected individuals and chains were formed. In the fif-

Heredity in Protozoa 619

teenth generation | began to save all the progeny of a; up to this
time only the specimen bearing the spine had been kept alive.
In the large number of progeny thus obtained many variants were
to be observed in the matter of interconnection. Many individ-
uals were free and separate. Pairs of united individuals were very
common. Chains of three to eight or more (Fig. 20) were not
uncommon. ‘These longer chains were likely to break apart in the
course of time, as a result of their bending and twisting move-
ments.

Fig. 20 Chains of individuals formed in the history of the race a, as a result of incomplete fission.

There was much variation in the extent and strength of the
union. Sometimes there resulted from the division of united indi-
viduals specimens that were quite free. The division of free
specimens often produced united pairs. In some cases the con-
necting band was very thick and strong, so as to hold the two
specimens inflexibly in various positions (Fig. 20, 6). In other
cases the fission was so incomplete that mere partly double
specimens resulted (parts of ¢, Fig. 20). Finally, the irregularities

620 H. S. fennings

of fission at times went so far as to produce mere monstrosities
(parts of d, Fig. 20). Such monstrosities were rare, while indi-
viduals neatly united in pairs or in chains were very common.

The first occurrence of such unions (Fig. 4, 1°) was on May to.
Cultures were kept in watch glasses from that time till July 1
(probably about fifty generations); at that date the unions were
still abundant. In fifty generations the original individual which
underwent the modification causing the union would have pro-
duced progeny to a number running far up into the billions.

Effects of Artificial Selection

On June 22 I began experiments to determine the effect of selec-
tion on this peculiarity. Would it be possible by selection to pro-
duce on the one hand a series showing little or no tendency to
remain united, on the other hand a series in which most or all
the individuals remain in united pairs ?

Two selected cultures were started in watch glasses. ‘The first
contained twenty individuals united two by two in ten pairs.
The second contained twenty free individuals (descended from
the same ancestors as the united pairs).

Forty-eight hours later (June 24), both sets had multiplied to
about 100 specimens. In the first set (ancestors united) there were
ten united pairs. In the second set (free ancestors) there were
two united pairs.

From the first set | removed all the free individuals, leaving only
the ten united pairs. From the second set the two united pairs
were removed, leaving all free.

The further history was as follows:

Culture from free ancestors. On June 25 this had multiplied
to 200-400; among these were three or four united pairs. I re-
moved the latter and retained only 100 of the free individuals.

On June 26 these had multiplied two to four times but contained
no united specimens. This culture was kept for a week or so
longer, but developed no more united pairs. Thus, selection had
quite removed from this set the tendency to remain united.

Culture from united ancestors. After the second isolation of ten
united pairs (June 24), the number multiplied to about 50 in 24

Heredity in Protozoa 621

hours; among these there were eight groups of united individ-
uals—some of two, some of several, specimens united in chains.
The eight groups were again isolated ( June 25).

Effects of Natural Selection

These eight groups showed many imperfect individuals, and
the groups were at a great disadvantage as compared with the free
individuals. ‘This was because they are not able to swim about
actively, but must lie at the bottom and move about only irregu-
larly. As a result they get comparatively little food, and are not
able to avoid regions where the conditions are harmful. The bac-
teria multiplied much more rapidly than in the free culture, con-
taining many individuals—the latter keeping down the number
of bacteria by feeding on them.

In consequence of these bad conditions, the united groups began
to die. Some multiplied farther, all the individuals remaining
united. But forty-eight hours after the isolation of the second lot
of eight groups, all were dead.

Thus it is easy to produce by selection a culture containing only
free individuals and multiplying in the usual way. Artificial
selection will likewise produce a culture of united specimens,
multiplying mainly by incomplete fission. But at the same time
natural selection acts; these groups die, owing to their inefliciency
in getting food, keeping down the bacteria, avoiding harm, and
in the performance of the general bodily functions.

This extinction by natural selection of the series multiplying
by incomplete fission was shown in another way. A considerable
number of the progeny of a, with both separated individuals and
united groups, was allowed to accumulate in a shallow watch glass.
Here the united groups flourished fairly well, because the vessel
was so shallow that they received plenty of oxygen and of food
while lying on the bottom, while the undue multiplication of the
bacteria was prevented by the numerous free individuals. Now
the culture was transferred to a large vessel, three inches deep.
Here the culture multiplied enormously, but all the groups of
united specimens quickly disappeared. “They sank to the bottom

622 lal S. ‘fennings

of the vessel, where the conditions were not such as to keep them
alive, while the free individuals remained at the top and multi-
plied. Thus by continued natural selection all specimens mul-
tiplying by incomplete fission were removed, and in a few days
the deep culture contained only normal, free individuals. In
shallow cultures, on the other hand, the united groups persisted
for about two months, as we have seen.

In this case then we have a new characteristic, of known origin,
that is inherited by many individuals for many generations, and is
finally extinguished only by the action of natural selection. The
many other new characteristics that we have described were not
inherited (save as they were handed on directly to a single speci-
men). In the one case the new feature becomes a race charac-
teristic; in all the others it fails to do so.

WHAT MUST BE THE NATURE OF -A NEW CHARACTER, THAT IT
MAY BE INHERITED?

What is the peculiarity of the characteristic that was thus mul-
tiplied and inherited, and what light does it throw on the question
as to what must be the nature of an acquired characteristic in order
that it may be inherited ?

The characteristic thus inherited was a modification of the pro-
toplasm of the cell, of such a character as to cause 1t to behave differ-
ently in reproduction. The other characteristics, not inherited,
were not such modifications of the protoplasm as to cause it to
behave differently in reproduction.

Consideration of the facts of normal reproduction in the Pro-
tozoa, and of heredity in general, indicates that this difference is an
essential one. In order that it may be inherited (by more than one
of the progeny), a characteristic must be the result of such a modi-
fication of the mother cell as will cause it to behave in a certain way
at reproduction. It makes no difference whether the mother cell
in question is a germ cell, in a Metazoan, or a differentiated Pro-
tozoan.

Thus we know that in the inheritance of the seta of the Hypo-
tricha, for example (Fig. 21), these are not simply handed over in

Heredity in Protozoa 623

finished form, like the spine of a (Fig. 4), but are produced anew
on each product of fission. The old seta and cilia degenerate and
disappear as fission sets in. Inthe daughter individuals the new
seta appear in a small group with a totally different arrangement
from that seen in the adult parent (Fig. 21, ») and the final arrange-
ment 1s reached by complicated processes of differentiation and
distribution. Thus the presence of setae in the posterity could
have been brought about in the beginning only by such modifica-
tions of the protoplasm of the mother cell as would cause it at fis-
sion to produce sete. Any change in the structure, number, or

Fig. 21 Dividing Stylonychia, from Biitschli, showing at « the appearance of the new sete in a close

group

arrangement of the setz could result only from such a modification
of the mother cell as would alter in a definite way the processes
occurring at reproduction. The thing transmitted from the
parent cell to the young progeny is, not the seta themselves, but
the change in the protoplasm causing the production of sete in a
definite way.

To return to a specific problem—How then could such a local-
ized appendage as the spine of a (Fig. 4) become an inherited char-
acteristic? Only through such a modification of the protoplasm

624 H. 8. “‘fennings

of the parent cell as would cause at fission the production of such
an appendage on each of the progeny.

At first thought it appears difficult to conceive how this could
occur. This will be made easier, perhaps, by a consideration
of the origin of certain characteristics in the race a (Fig. 4, etc.).

Examples of Modifications from which New Inherited Charac-
ters Might Result

Let us take first the origin of the spine whose history is traced
in Fig. 4. The original ancestor of the race a was without spines.
But it was so deformed and modified that at the time of fission
two short teeth were produced during the processes of division
(Fig. 3,7). At the next fission one of these short teeth formed as
it were a region of weakness, where a long spine was pushed out,
as an accompaniment of the processes of fission (Fig. 3, * °).
Such a region of weakness might well exist without a visible tooth
to show its position; then at fission a spine would be produced in
this spot. It is evident that active physical and chemical processes
are in progress at the time of fission; these may easily result, under
the influence of a local modification of the parent cell, in the push-
ing out of a spine or other’structure of characteristic form.

How such a new structure might appear in each of the progeny
of each generation is illustrated in a simple way by certain other
phenomena seen in the race a. As we have already set forth, the
progeny of a showed after a certain period a tendency to remain
united in chains. At the same time there appeared among the
free progeny of a a considerable number of individuals which bore
at one or both ends a spike-like point (Fig. 22). This character
did not become general, but so many cases appeared that one
might say that there was an inherited tendency toward this. Ob-
servation of the process of fission showed that these points arose
by the pulling out of the protoplasm while in the plastic condition
at the time of fission; the two young animals were connected, at a
certain stage, by a bridge of this plastic protoplasm. By their
movements they drew this out to a long strand, which finally broke
at the middle, leaving a point at the ends of the two animals.

Heredity in Protozoa 625

When this happened at successive fissions, the animal bore such
a point at each end.

It is evident that these points are due to the same cause that pro-
duced the inherited tendency to remain united in chains (as in Fig.
20). They result from the ridge of new material along the aboral
side of the animal, shown in Fig. 4, °. Now, it is easily conceiv-
able that this new material might be of such texture and thickness
that it would always be drawn out at fission in such a way as to
produce points of a definite form and size. These would then
appear regularly after fission; a race of Paramecia with this as

Fig. 22 Examples from the race a, of individuals having a point at the posterior end, due to the draw-
ing out of the connecting band at the time of fission.

a new characteristic would have been produced. ‘The spine would
be hereditary, because produced anew in each generation, just
as are the setae of the Hypotricha, or the organs of the Metazoa*

SUMMARY AND GENERAL DISCUSSION

The following general statements of the laws and principles
bearing on heredity} that result from our investigation are made
with direct reference to the Protozoa, and will best be grasped
by keeping in mind concrete cases, such for example as those
shown in Fig. 4, Fig. 20 and Fig. 22.

*It is of course possible that the origin of new permanently inherited characters is not normally through
mere modifications of the external parts of the cell, such as we see in our illustrative cases. Possibly
there must be originally some modification of more recondite parts—nucleus, chromosomes, or the like—
and that these then secondarily act upon and change the outer parts. This would add farther compli-
cation, but would not change the essential point, which is, that in order that a characteristic may be
inherited, it must be due to some modification that causes a change in the processes of reproduction.

+For a summary of results on other matters than heredity (on the changes during fission and growth,

etc.), see pp. 599-604.

626 Jake S. fennings

1 The “inheritance of acquired characters’ meets the same
difficulty in the Protozoa as in Metazoa. In both Protozoa and
Metazoa most characteristics acquired during the lifetime of the
individual are not inherited, and such inheritance does not occur
more readily in the one group than in the other.

2 The difficulty with the “inheritance of acquired characters”
lies, not in the separation of soma and germ, but in the process
of cell division. Ifa cell bears a structure at one end, there is no
simple and direct reason why, when it divides, both the cells pro-
duced should bear the structure, and observation shows that they
do not, in the case of new structures. ‘There is no evident way in
which a structure of this sort can overleap the barrier of cell divi-
sion and appear on the other side.*

If we insist on making a comparison between the condition in
the Protozoa and the separation of soma and germ in the Metazoa,
the following is the state of the case. If any Protozoan cell (as
in Fig. 7) is to be divided at the next fission into two parts a and p,
then, so far as inheritance of new structures is concerned, a stands
to p as soma to germ, and reciprocally, p stands to a as soma to
germ. In other words, there is no evident transmission, and no
evident mechanism for transmission, of a new structure from a
to p or the reverse, just as there is no evident mechanism for trans-
mitting a structure from soma to germ.

3. In order that a character may be inherited (by more than
one of the progeny, so as to produce a race), it must be produced
anew in each generation. ‘This is what happens in the normal
reproduction of both Protozoa and Metazoa.

4 In order that a new (or “acquired”’) character may be
inherited, it must be the result of such a modification of the parent
cell as will cause a change in the processes of reproduction; and
specifically, precisely such a change in these processes as will pro-
duce the character in question. ‘This is equally true of Protozoa
and Metazoa.

5 Most characteristics acquired during the life-time of the

* This will be most readily grasped by looking at the figure of a typical case, such as Fig. 4,%. Why,

when this animal divides transversely, should there be a spine upon the posterior (left) half, as well as
upon the anterior (right)? As a matter of fact, there is mor.

Heredity in Protozoa 627

individual are not the result of such modifications of the parent
cell as will cause a change in the process of reproduction such
as to produce anew these characteristics; hence they ar: not
inherited. This is true in both Protozoa and Metazoa.

6 Thus the problem of how new inherited characters arise is
the same in Protozoa as in Metazoa. We may therefore work
on the general problem as readily in the one group as in the other,
and there is no reason why the principles reached in one group
should not apply equally to the other. Thus a new line of attack
on the problem is opened; in view of the rapid multiplication of
the Protozoa and the ready accessybility of their reproductive cells
both to environmental influences and to observation, this gives
some marked advantages.

7 The search for the origin of new inherited characters (in both
Protozoa and Metazoa) resolves itself experimentally into a search
for agencies and processes which will permanently modify the
cell in such a way as to cause it to act differently in reproduction.

8 When a given structural characteristic arises during the
reproductive processes so as to appear in a given generation, that
is not because the same structlire was present in a preceding g gen-
eration. Often indeed it was not present before; its origin is due
to some change in the constitution (chemical or structural?) of
the preceding reproductive cell. Thus, the production of a spine
such as we see in Fig. 4 is evidently due to a spot of weakness at a
certain point in the cell body, causing a protrusion during fission.
Such a structure might result from the localized presence some-
where in the cell body of a certain chemical compound, which
would react at a certain stage with some other substance, thus
producing a spot of weakness, where a spine would be protruded.
So, the appearance of the new anterior sete in the posterior prod-
uct of division in the Hypotricha (Fig. 21) is evidently due in
some way to the constitution of the cell.

9g Thus, then, the cause of the appearance of a certain struc-
ture in a certain generation is some other peculiarity of the cell
producing it; some chemical peculiarity, for example. We may
generalize this by saying that the appearance in the progeny of
a certain structure } is due to the existence in the mother cell of a
quite different condition a.

628 Jala We ‘fennings

10 It follows from what has been set forth in the paragraphs
preceding, that in the production of a new inherited character the
original modification will be something quite different from the
visible structural characteristic which later appears in consequence
of it. The original modification will be some chemical or struc-
tural change in the reproductive cell or cells that are later to pro-
duce the structure in question. (By producing in Paramecium
a localized change in the character of the protoplasm, a spine is
later produced at that spot, etc.) The first appearance of the
visible structure is one generation after the production of the modifi-
cation to which it is due.

11 Not all modifications of the germ cells that result in the
production of a new character in the next generation, will result
in the repeated production of this character in succeeding genera-
tions. In most cases, the new structure appears but once, and is
not inherited. In order that the new structure shall be inherited,
the original modification to which it 1s due must be transmitted
to the succeeding generation of germ cells. This is by no means
a matter of course; in fact, it is something not to be expected, as a
rule. The cell usually, by regulative processes, throws off after
a time any modification which the environment has impressed
upon it. Many examples of this are seen in the foregoing pages.
Certain unusual conditions of the cell result in the production, at
the next fission, of a spine. But during fission regulation occurs;
the unusual condition disappears, and the spine is not again pro-
duced.

This is doubtless the fate of most modifications of the cell. We
saw, however, one modification which persisted, producing its
effect in succeeding generations (pp. 618-622). Of such a nature
must be all modifications which produce new inherited character-
istics. It 1s easy to so modify the cell that new characteristics shall
appear in one succeeding generation; to so modify it that the new
characteristic shall appear regularly in succeeding generations is a
totally different matter.

We often hear it pointed out that heredity is not transmission,
but new production; and this has been emphasized in the pre-
ceding pages. But it needs to be realized that while it is true that

Heredity in Protozoa 629

the inherited structure visibly appearing is not transmitted, sore-
thing is transmitted, namely, the condition of the protoplasm
which causes the production of the visible inherited structure. If
this determining condition were not transmitted, the visible
structure could not be produced in each generation. It is this
“something” transmitted that lies at the basis of the figurative
expression “bearer of heredity,” or the like.

12 What sort of modifications will remain permanently and be
transmitted to the progeny? Evidently, only such modifications
as are not removed by the regulatory processes of the cell. The
modifications that are removed by regulation are precisely those
which interfere in one way or another with the physiological proc-
esses of the organism, while modifications which arise in harmony
with, or as a result of, the normal functioning of the cell are not
removed by regulation. Thus only characteristics of the latter
class—namely, adaptive characteristics—will be retained and
transmitted. Furthermore, it appears clear that the successive
modifications in the reproductive processes induced by these
adaptive characteristics must likewise be in harmony with the
normal functioning of the cell, else they would be removed by the
known regulatory activities of the cell. Thus all stages in the
modification, including the final one, must be in harmonious
adjustment to the normal activities of the organism. It would
appear therefore that only the new characteristics that are adaptive
will be inherited. Anything not in harmony with the normal func-
tioning of the cell will be removed by regulation.

13 Let us now examine the problem of the “inheritance of
acquired characters.” What processes would be required for the
inheritance by the progeny of the same characteristic that has
already been produced directly in the parent, by environmental
action ?

Keeping the Protozoa in mind, we have evidently two cases
here:

a If the “acquired character” is some general chemical or
structural change in the parent cell—something that affects the
cell as a whole—then there appears to be no special difficulty in
the way of a direct transmission of this to the progeny, provided

630 al. S. fennings

it is not thrown off by regulation. _ If new inherited characters of
any sort are ever produced by environmental action, such direct
transmission of an acquired internal modification must occur, as
we have already seen (paragraph 11). In the Metazoa, it would
evidently be only general changes in the germ cells that would be
thus directly transmitted.

b The case of a new /ocalized modification or of a definite new
structure, such as a spine, which is directly produced by environ-
mental action, is wholly different. As we have already seen (para-
graphs g, 10, 11), in order that a new localized structure 4 shall
appear in each generation, a certain other condition a must be
produced in the mother cells; this condition a must be transmitted
from generation to generation, and must so modify the reproductive
processes as to cause, at each fission, the production of the new
structure b.

Now, if the new structure b was first produced directly in the
parent by environmental action, and is then to be inherited, the
processes required are the following. The existence of the struc-
ture ) (a spine, for example), in the parent cell, must cause the
production in that parent cell of precisely the “other” condition
a, that is of such a nature as to so change the processes of repro-
duction that they will again produce identically the character }
(the spine) which had first been produced by the environment.
Or, what amounts to practically the same thing, the environment
must coincidentally produce two heterogeneous effects: (1) it
must directly produce the structure b; (2) it must produce some
permanent change a in the constitution of the cell, such as_ will so
modify the processes of reproduction that they in their turn will
produce the same structure 6:

Such coincidental production of a complex structure b in two
quite heterogeneous ways would be most extraordinary, and we
have as yet no glimmering of a mechanism by which the coinci-
dence could be produced. Moreover, as we have seen, in most
cases (in all precisely observed cases) it is not produced; we have
little if any direct evidence that it ever occurs.

Yet if it ever occurred it would be of such importance that we
must of course continue to be on the watch, in all experimental

Heredity in Protozoa 631

work, for any evidence of it. The question, put as simply as
possible, is as follows:

Is there ever any mechanism or property in virtue of which,
when a structural modification occurs in one part of the body,
this will modify another part of the body (not in the same way, but)
in such a way that this other part will, at reproduction, start up
processes tending to produce a similar structural modification ?

14 The propositions thus far set forth have had direct refer-
ence to the Protozoa; but in the main they apply a fortror: to the
Metazoa also. The difference between the two groups as to
heredity is not one of principle, but of complexity. The extreme
difference in complexity may be put as follows:

In the Protozoa, when a new inherited character is to appear
in the adult, this requires a modification of the adult of the pre-
vious generation, of such a character as to change in a definite
way only the next fission and processes immediately connected
with it. This requirement is sufficiently complex when we come
to ask how the numerous locomotor organs of the Hypotricha, in
their varied typical patterns, have arisen and become hereditary.
But it is not to be compared in complexity with what we have to
set forth next.

In the Metazoa the requirement for the sppeotanes of a heredi-
tary new structure in the adult is that the preceding germ cell
shall be so modified that at the next fission the reproductive proc-
esses shall be changed, but the change shall not yet be of a char-
acter to produce the ultimate structures. In the next and the
next, and in hundreds of succeeding fissions the processes must all
be modified so as to keep in each cell the conditions for the final
production of the ultimate new structure. These conditions will
necessarily be different in the different cell generations, as differ-
entiation occurs, and of course each of the intermediate condi-
tions is something quite diverse from the final structure. At the
end the new structure is produced, not by a modification in the
re productive processes of one cell, as in the Protozoa, nor by the
same modifications in many cells, but by the diverse modifications
of thousands and thousands of cells, all so modified as to cooperate
in the production of the final structure. The mind refuses the
useless attempt to conceive of such complexity of change.

622 H. 8. fennings

As Conklin (’08) has so well set forth in a recent address,
“the mechanism of heredity is merely the mechanism of differen-
tiation.” The questions with which we have to deal are those as

to the nature of the determining conditions and of the processes,

by which the constitution of the cell changes. Perhaps the most
direct study of heredity possible in the Metazoa is such a study as
Conklin is making of the internal determining conditions in the
differentiating cells of the developing organism. When one comes
to the study of heredity in the Protozoa, this simply coincides with
a study of the determining causes of differentiation.

Johns Hopkins University
Baltimore, Md.
March 10, 1908

LITERATURE CITED

Carkins, G. N. ’02—Studies on the life history of Protozoa. I. The life history
of Paramcecium caudatum. Arch. f. Entw.-mech., 15, 139-186.
’06—The Protozoan life cycle. Biol. Bul., x1, 229-244.

Conkun, E. G. ’08—The mechanism of heredity. Science, xxvil, 89-99.

Jenninas, H. S. ’06—Behavior of the lower organisms. 366 pp. New York.

Jensen, P. ’07—Organische Zweckmissigkeit, Entwicklung und Vererbung vom
Standpunkt der Physiologie. 251 pp. Jena.

Kettoce, V. L.’07—Darwinism today. 403 pp. New York.

Roux, W. ’81—Der Kampf der Theile im Organismus. Leipzig.

Tower, W. L.’06—An investigation of evolution in chrysomelid beetles of the genus
Leptinotarsa. Carnegie Inst. of Washington, Pub. 26, 320 pp.

Wurman, C. O. ’99—Animal behavior. Woods Hole Biol. Lectures for 1898, pp.

285-338.

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